Nucleic acids encoding a G-protein coupled receptor involved in sensory transduction

ABSTRACT

The invention provides isolated nucleic acid and amino acid sequences of sensory cell specific G-protein coupled receptors, antibodies to such receptors, methods of detecting such nucleic acids and receptors, and methods of screening for modulators of sensory cell specific G-protein coupled receptors.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 09/361,631, filedJul. 27, 1999, now U.S. Pat. No. 6,383,778, which claims priority toU.S. Ser. No. 60/095,464, filed Jul. 28, 1998, and U.S. Ser. No.60/112,747, filed Dec. 17, 1998, each herein incorporated by referencein its entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with government support under Grant No. 5R01DC03160, awarded by the National Institutes of Health. The governmenthas certain rights in this invention.

FIELD OF THE INVENTION

The invention provides isolated nucleic acid and amino acid sequences ofsensory cell specific G-protein coupled receptors, antibodies to suchreceptors, methods of detecting such nucleic acids and receptors, andmethods of screening for modulators of sensory cell specific G-proteincoupled receptors.

BACKGROUND OF THE INVENTION

Taste transduction is one of the most sophisticated forms ofchemotransduction in animals (see, e.g., Margolskee, BioEssays15:645–650 (1993); Avenet & Lindemann, J. Membrane Biol. 112:1–8(1989)). Gustatory signaling is found throughout the animal kingdom,from simple metazoans to the most complex of vertebrates; its mainpurpose is to provide a reliable signaling response to non-volatileligands. Each of these modalities is though to be mediated by distinctsignaling pathways mediated by receptors or channels, leading toreceptor cell depolarization, generation of a receptor or actionpotential, and release of neurotransmitter at gustatory afferent neuronsynapses (see, e.g., Roper, Ann. Rev. Neurosci. 12:329–353 (1989)).

Mammals are believed to have five basic taste modalities: sweet, bitter,sour, salty and unami (the taste of monosodium glutamate) (see, e.g.,Kawamura & Kare, Introduction to Unami: A Basic Taste (1987); Kinnamon &Cummings, Ann. Rev. Physiol. 54:715–731(1992); Lindemann, Physiol. Rev.76:718–766 (1996); Stewart et al., Am. J. Physiol. 272:1–26 (1997)).Extensive psychophysical studies in humans have reported that differentregions of the tongue display different gustatory preferences (see,e.g., Hoffmann, Menchen. Arch. Path. Anat. Physiol. 62:516–530 (1875);Bradley et al., Anatomical Record 212: 246–249 (1985); Miller & Reedy,Physiol. Behav. 47:1213–1219 (1990)). Also, numerous physiologicalstudies in animals have shown that taste receptor cells may selectivelyrespond to different tastants (see, e.g., Akabas et al., Science242:1047–1050 (1988); Gilbertson et al., J. Gen. Physiol. 100:803–24(1992); Bernhardt et al., J. Physiol. 490:325–336 (1996); Cummings etal., J. Neurophysiol. 75:1256–1263 (1996)).

In mammals, taste receptor cells are assembled into taste buds that aredistributed into different papillae in the tongue epithelium.Circumvallate papillae, found at the very back of the tongue, containhundreds (mice) to thousands (human) of taste buds and are particularlysensitive to bitter substances. Foliate papillae, localized to theposterior lateral edge of the tongue, contain dozens to hundreds oftaste buds and are particularly sensitive to sour and bitter substances.Fungiform papillae containing a single or a few taste buds are at thefront of the tongue and are thought to mediate much of the sweet tastemodality.

Each taste bud, depending on the species, contain 50–150 cells,including precursor cells, support cells, and taste receptor cells (see,e.g., Lindemann, Physiol. Rev. 76:718–766 (1996)). Receptor cells areinnervated at their base by afferent nerve endings that transmitinformation to the taste centers of the cortex through synapses in thebrain stem and thalamus. Elucidating the mechanisms of taste cellsignaling and information processing is critical for understanding thefunction, regulation, and “perception” of the sense of taste.

Although much is known about the psychophysics and physiology of tastecell function, very little is known about the molecules and pathwaysthat mediate these sensory signaling responses (reviewed by Gilbertson,Current Opn. in Neurobiol 3:532–539 (1993)). Electrophysiologicalstudies suggest that sour and salty tastants modulate taste cellfunction by direct entry of H⁺ and Na⁺ ions through specialized membranechannels on the apical surface of the cell. In the case of sourcompounds, taste cell depolarization is hypothesized to result from H⁺blockage of K⁺ channels (see, e.g., Kinnamon et al., Proc. Nat'l Acad.Sci. USA 85: 7023–7027 (1988)) or activation of pH-sensitive channels(see, e.g., Gilbertson et al., J. Gen. Physiol. 100:803–24 (1992)); salttransduction may be partly mediated by the entry of Na⁺ viaamiloride-sensitive Na⁺ channels (see, e.g., Heck et al., Science223:403–405 (1984); Brand et al., Brain Res. 207–214 (1985); Avenet eta., Nature 331: 351–354 (1988)).

Sweet, bitter, and unami transduction are believed to be mediated byG-protein-coupled receptor (GPCR) signaling pathways (see, e.g., Striemet al., Biochem. J. 260:121–126 (1989); Chaudhari et al., J. Neuros.16:3817–3826 (1996); Wong et al., Nature 381: 796–800 (1996)).Confusingly, there are almost as many models of signaling pathways forsweet and bitter transduction as there are effector enzymes for GPCRcascades (e.g., G protein subunits, cGMP phosphodiesterase,phospholipase C, adenylate cyclase; see, e.g., Kinnamon & Margolskee,Curr. Opin. Neurobiol. 6:506–513 (1996)). However, little is known aboutthe specific membrane receptors involved in taste transduction, or manyof the individual intracellular signaling molecules activated by theindividual taste transduction pathways. Identification of such moleculesis important given the numerous pharmacological and food industryapplications for bitter antagonists, sweet agonists, and modulators ofsalty and sour taste.

The identification and isolation of taste receptors (including taste ionchannels), and taste signaling molecules, such as G-protein subunits andenzymes involved in signal transduction, would allow for thepharmacological and genetic modulation of taste transduction pathways.For example, availability of receptor and channel molecules would permitthe screening for high affinity agonists, antagonists, inverse agonists,and modulators of taste cell activity. Such taste modulating compoundscould then be used in the pharmaceutical and food industries tocustomize taste. In addition, such taste cell specific molecules canserve as invaluable tools in the generation of taste topographic mapsthat elucidate the relationship between the taste cells of the tongueand taste sensory neurons leading to taste centers in the brain.

SUMMARY OF THE INVENTION

The present invention thus provides for the first time nucleic acidsencoding a taste cell specific G-protein coupled receptor. These nucleicacids and the polypeptides that they encode are referred to as “GPCR-B4”for G-protein coupled receptor (“GPCR”) B4. These taste cell specificGPCRs are components of the taste transduction pathway.

In one aspect, the present invention provides an isolated nucleic acidencoding a sensory transduction G-protein coupled receptor, the receptorcomprising greater than about 70% amino acid identity to an amino acidsequence of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:7.

In one embodiment, the nucleic acid comprises a nucleotide sequence ofSEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:8. In another embodiment, thenucleic acid is amplified by primers that selectively hybridize understringent hybridization conditions to the same sequence as degenerateprimer sets encoding amino acid sequences selected from the groupconsisting of: SAGGPMCFLM (SEQ ID NO:5) and WMRYHGPYVF (SEQ ID NO:6).

In another aspect, the present invention provides an isolated nucleicacid encoding a sensory transduction G-protein coupled receptor, whereinthe nucleic acid specifically hybridizes under highly stringentconditions to a nucleic acid having the sequence of SEQ ID NO:3, SEQ IDNO:4, or SEQ ID NO:8.

In another aspect, the present invention provides an isolated nucleicacid encoding a sensory transduction G-protein coupled receptor, thereceptor comprising greater than about 70% amino acid identity to apolypeptide having a sequence of SEQ ID NO:1, SEQ ID NO:2, or SEQ IDNO:7, wherein the nucleic acid selectively hybridizes under moderatelystringent hybridization conditions to a nucleotide sequence of SEQ IDNO:3, SEQ ID NO:4, or SEQ ID NO:8.

In another aspect, the present invention provides an isolated nucleicacid encoding an extracellular domain of a sensory transductionG-protein coupled receptor, the extracellular domain having greater thanabout 70% amino acid sequence identity to the extracellular domain ofSEQ ID NO:1.

In another aspect, the present invention provides an isolated nucleicacid encoding a transmembrane domain of a sensory transduction G-proteincoupled receptor, the transmembrane domain comprising greater than about70% amino acid sequence identity to the transmembrane domain of SEQ IDNO:1.

In another aspect, the present invention provides an isolated sensorytransduction G-protein coupled receptor, the receptor comprising greaterthan about 70% amino acid sequence identity to an amino acid sequence ofSEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:7.

In one embodiment, the receptor specifically binds to polyclonalantibodies-generated against SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:7.In another embodiment, the receptor has G-protein coupled receptoractivity. In another embodiment, the receptor has an amino acid sequenceof SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:7. In another embodiment, thereceptor is from a human, a rat, or a mouse.

In one aspect, the present invention provides an isolated polypeptidecomprising an extracellular domain of a sensory transduction G-proteincoupled receptor, the extracellular domain comprising greater than about70% amino acid sequence identity to the extracellular domain of SEQ IDNO:1.

In one embodiment, the polypeptide encodes the extracellular domain ofSEQ ID NO:1. In another embodiment, the extracellular domain iscovalently linked to a heterologous polypeptide, forming a chimericpolypeptide.

In one aspect, the present invention provides an isolated polypeptidecomprising a transmembrane domain of a sensory transduction G-proteincoupled receptor, the transmembrane domain comprising greater than about70% amino acid sequence identity to the transmembrane domain of SEQ IDNO:1.

In one embodiment, the polypeptide encodes the transmembrane domain ofSEQ ID NO:1. In another embodiment, the polypeptide further comprises acytoplasmic domain comprising greater than about 70% amino acid identityto the cytoplasmic domain of SEQ ID NO:1. In another embodiment, thepolypeptide encodes the cytoplasmic domain of SEQ ID NO:1. In anotherembodiment, the transmembrane domain is covalently linked to aheterologous polypeptide, forming a chimeric polypeptide. In anotherembodiment, the chimeric polypeptide has G-protein coupled receptoractivity.

In one aspect, the present invention provides an antibody thatselectively binds to the receptor comprising greater than about 70%amino acid sequence identity to an amino acid sequence of SEQ ID NO:1,SEQ ID NO:2, or SEQ ID NO:7.

In another aspect, the present invention provides an expression vectorcomprising a nucleic acid encoding a polypeptide comprising greater thanabout 70% amino acid sequence identity to an amino acid sequence of SEQID NO:1, SEQ ID NO:2, or SEQ ID NO:7.

In another aspect, the present invention provides a host celltransfected with the expression vector.

In another aspect, the present invention provides a method foridentifying a compound that modulates sensory signaling in sensorycells, the method comprising the steps of: (i) contacting the compoundwith a polypeptide comprising an extracellular domain of a sensorytransduction G-protein coupled receptor, the extracellular domaincomprising greater than about 70% amino acid sequence identity to theextracellular domain of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:7; and(ii) determining the functional effect of the compound upon theextracellular domain.

In another aspect, the present invention provides a method foridentifying a compound that modulates sensory signaling in sensorycells, the method comprising the steps of: (i) contacting the compoundwith a polypeptide comprising an extracellular domain of a sensorytransduction G-protein coupled receptor, the transmembrane domaincomprising greater than about 70% amino acid sequence identity to theextracellular domain of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:7; and(ii) determining the functional effect of the compound upon thetransmembrane domain.

In one embodiment, the polypeptide is a sensory transduction G-proteincoupled receptor, the receptor comprising greater than about 70% aminoacid identity to a polypeptide encoding SEQ ID NO:1, SEQ ID NO:2, or SEQID NO:7. In another embodiment, polypeptide comprises an extracellulardomain that is covalently linked to a heterologous polypeptide, forminga chimeric polypeptide. In another embodiment, the polypeptide hasG-protein coupled receptor activity. In another embodiment, theextracellular domain is linked to a solid phase, either covalently ornon-covalently. In another embodiment, the functional effect isdetermined by measuring changes in intracellular cAMP, IP3, or Ca²⁺. Inanother embodiment, the functional effect is a chemical effect. Inanother embodiment, the functional effect is a chemical effect. Inanother embodiment, the functional effect is determined by measuringbinding of the compound to the extracellular domain. In anotherembodiment, the polypeptide is recombinant. In another embodiment, thepolypeptide is expressed in a cell or cell membrane. In anotherembodiment, the cell is a eukaryotic cell.

In one embodiment, the polypeptide comprises an transmembrane domainthat is covalently linked to a heterologous polypeptide, forming achimeric polypeptide.

In one aspect, the present invention provides a method of making asensory transduction G-protein coupled receptor, the method comprisingthe step of expressing the receptor from a recombinant expression vectorcomprising a nucleic acid encoding the receptor, wherein the amino acidsequence of the receptor comprises greater than about 70% amino acididentity to a polypeptide having a sequence of SEQ ID NO:1, SEQ ID NO:2,or SEQ ID NO:7.

In one aspect, the present invention provides a method of making arecombinant cell comprising a sensory transduction G-protein coupledreceptor, the method comprising the step of transducing the cell with anexpression vector comprising a nucleic acid encoding the receptor,wherein the amino acid sequence of the receptor comprises greater thanabout 70% amino acid identity to a polypeptide having a sequence of SEQID NO:1, SEQ ID NO:2, or SEQ ID NO:7.

In one aspect, the present invention provides a method of making anrecombinant expression vector comprising a nucleic acid encoding asensory transduction G-protein coupled receptor, the method comprisingthe step of ligating to an expression vector a nucleic acid encoding thereceptor, wherein the amino acid sequence of the receptor comprisesgreater than about 70% amino acid identity to a polypeptide having asequence of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:7.

BRIEF DESCRIPTION OF THE DRAWINGS

Not applicable.

DETAILED DESCRIPTION OF THE INVENTION

I. Introduction

The present invention provides for the first time nucleic acids encodinga taste cell specific G-protein coupled receptor. These nucleic acidsand the receptors that they encode are referred to as “GPCR” forG-protein coupled receptor, and are designated as GPCR-B4. These tastecell specific GPCR are components of the taste transduction pathway(see, e.g., Example II). These nucleic acids provide valuable probes forthe identification of taste cells, as the nucleic acids are specificallyexpressed in taste cells. For example, probes for GPCR polypeptides andproteins can be used to identity subsets of taste cells such as foliatecells and circumvallate cells, or specific taste receptor cells, e.g.,sweet, sour, salty, and bitter. They also serve as tools for thegeneration of taste topographic maps that elucidate the relationshipbetween the taste cells of the tongue and taste sensory neurons leadingto taste centers in the brain. Furthermore, the nucleic acids and theproteins they encode can be used as probes to dissect taste-inducedbehaviors.

The invention also provides methods of screening for modulators, e.g.,activators, inhibitors, stimulators, enhancers, agonists, andantagonists, of these novel taste cell GPCRs. Such modulators of tastetransduction are useful for pharmacological and genetic modulation oftaste signaling pathways. These methods of screening can be used toidentify high affinity agonists and antagonists of taste cell activity.These modulatory compounds can then be used in the food andpharmaceutical industries to customize taste. Thus, the inventionprovides assays for taste modulation, where GPCR-B4 acts as an direct orindirect reporter molecule for the effect of modulators on tastetransduction. GPCRs can be used in assays, e.g., to measure changes inion concentration, membrane potential, current flow, ion flux,transcription, signal transduction, receptor-ligand interactions, secondmessenger concentrations, in vitro, in vivo, and ex vivo. In oneembodiment, GPCR-B4 can be used as an indirect reporter via attachmentto a second reporter molecule such as green fluorescent protein (see,e.g., Mistili & Spector, Nature Biotechnology 15:961–964 (1997)). Inanother embodiment, GPCR B-4s are recombinantly expressed in cells, andmodulation of taste transduction via GPCR activity is assayed bymeasuring changes in Ca²⁺ levels (see Example II).

Methods of assaying for modulators of taste transduction include invitro ligand binding assays using GPCR-B4, portions thereof such as theextracellular domain, or chimeric proteins comprising one or moredomains of GPCR-B4, oocyte GPCR-B4 expression; tissue culture cellGPCR-B4 expression; transcriptional activation of GPCR-B4;phosphorylation and dephosphorylation of GPCRs; G-protein binding toGPCRs; ligand binding assays; voltage, membrane potential andconductance changes; ion flux assays; changes in intracellular secondmessengers such as cAMP and inositol triphosphate; changes inintracellular calcium levels; and neurotransmitter release.

Finally, the invention provides for methods of detecting GPCR-B4 nucleicacid and protein expression, allowing investigation of tastetransduction regulation and specific identification of taste receptorcells. GPCR-B4 also provides useful nucleic acid probes for paternityand forensic investigations. GPCR-B4 is useful as a nucleic acid probefor identifying subpopulations of taste receptor cells such as foliate,fungiform, and circumvallate taste receptor cells. GPCR-B4 receptors canalso be used to generate monoclonal and polyclonal antibodies useful foridentifying taste receptor cells. Taste receptor cells can be identifiedusing techniques such as reverse transcription and amplification ofmRNA, isolation of total RNA or poly A⁺ RNA, northern blotting, dotblotting, in situ hybridization, RNase protection, S1 digestion, probingDNA microchip arrays, western blots, and the like.

Functionally, GPCR-B4 represents a seven transmembrane G-protein coupledreceptor involved in taste transduction, which interacts with aG-protein to mediate taste signal transduction (see, e.g., Fong, CellSignal 8:217 (1996); Baldwin, Curr. Opin. Cell Biol. 6:180 (1994)).

Structurally, the nucleotide sequence of GPCR-B4 (see, e.g., SEQ IDNOS:3–4 and 8, isolated from rat, mouse, and human respectively) encodesa polypeptide of approximately 842 amino acids with a predictedmolecular weight of approximately 97 kDa and a predicted range of 92–102kDa (see, e.g., SEQ ID NOS:1–2 and 7, isolated from rat, mouse, andhuman). Related GPCR-B4 genes from other species share at least about70% amino acid identity over a amino acid region at least about 25 aminoacids in length, optionally 50 to 100 amino acids in length. GPCR-B4 isspecifically expressed in foliate and fungiform cells, with lowerexpression in circumvallate taste receptor cells of the tongue. GPCR-B4is an moderately rare sequence found in approximately 1/150,000 cDNAsfrom an oligo-dT primed circumvallate cDNA library (see Example I).

The present invention also provides polymorphic variants of the GPCR-B4depicted in SEQ ID NO:1: variant #1, in which an isoleucine residue issubstituted for a leucine acid residue at amino acid position 8; variant#2, in which an aspartic acid residue is substituted for a glutamic acidresidue at amino acid position 26; and variant #3, in which a glycineresidue is substituted for an alanine residue at amino acid position 46.

Specific regions of the GPCR-B4 nucleotide and amino acid sequence maybe used to identify polymorphic variants, interspecies homologs, andalleles of GPCR-B4. This identification can be made in vitro, e.g.,under stringent hybridization conditions or PCR (using primers encodingSEQ ID NOS:5–6) and sequencing, or by using the sequence information ina computer system for comparison with other nucleotide sequences.Typically, identification of polymorphic variants and alleles of GPCR-B4is made by comparing an amino acid sequence of about 25 amino acids ormore, e.g., 50–100 amino acids. Amino acid identity of approximately atleast 70% or above, optionally 80% or 90–95% or above typicallydemonstrates that a protein is a polymorphic variant, interspecieshomolog, or allele of GPCR-B4. Sequence comparison can be performedusing any of the sequence comparison algorithms discussed below.Antibodies that bind specifically to GPCR-B4 or a conserved regionthereof can also be used to identify alleles, interspecies homologs, andpolymorphic variants.

Polymorphic variants, interspecies homologs, and alleles of GPCR B4 areconfirmed by examining taste cell specific expression of the putativeGPCR-B4 polypeptide. Typically, GPCR-B4 having the amino acid sequenceof SEQ ID NO:1–2 or 7 is used as a positive control in comparison to theputative GPCR-B4 protein to demonstrate the identification of apolymorphic variant or allele of GPCR-B4. The polymorphic variants,alleles and interspecies homologs are expected to retain the seventransmembrane structure of a G-protein coupled receptor.

GPCR-B4 nucleotide and amino acid sequence information may also be usedto construct models of taste cell specific polypeptides in a computersystem. These models are subsequently used to identify compounds thatcan activate or inhibit GPCR-B4. Such compounds that modulate theactivity of GPCR B4 can be used to investigate the role of GPCR-B4 intaste transduction.

The isolation of GPCR-B4 for the first time provides a means forassaying for inhibitors and activators of G-protein coupled receptortaste transduction. Biologically active GPCR-B4 is useful for testinginhibitors and activators of GPCR-B4 as taste transducers using in vivoand in vitro expression that measure, e.g., transcriptional activationof GPCR-B4; ligand binding; phosphorylation and dephosphorylation;binding to G-proteins; G-protein activation; regulatory moleculebinding; voltage, membrane potential and conductance changes; ion flux;intracellular second messengers such as cAMP and inositol triphosphate;intracellular calcium levels; and neurotransmitter release. Suchactivators and inhibitors identified using GPCR-B4, can be used tofurther study taste transduction and to identify specific taste agonistsand antagonists. Such activators and inhibitors are useful aspharmaceutical and food agents for customizing taste.

Methods of detecting GPCR B4 nucleic acids and expression of GPCR-B4 arealso useful for identifying taste cells and creating topological maps ofthe tongue and the relation of tongue taste receptor cells to tastesensory neurons in the brain. Chromosome localization of the genesencoding human GPCR-B4 can be used to identify diseases, mutations, andtraits caused by and associated with GPCR-B4.

II. Definitions

As used herein, the following terms have the meanings ascribed to themunless specified otherwise.

“Taste receptor cells” are neuroepithelial cells that are organized intogroups to form taste buds of the tongue, e.g., foliate, fungiform, andcircumvallate cells (see, e.g., Roper et al., Ann. Rev. Neurosci.12:329–353 (1989)).

“GPCR-B4,” also called “TR2,” refers to a G-protein coupled receptorthat is specifically expressed in taste receptor cells such as foliate,fungiform, and circumvallate cells (see, e.g., Hoon et al., Cell96:541–551 (1999), herein incorporated by reference in its entirety).Such taste cells can be identified because they express specificmolecules such as Gustducin, a taste cell specific G protein (McLaughinet al., Nature 357:563–569 (1992)). Taste receptor cells can also beidentified on the basis of morphology (see, e.g., Roper, supra). GPCR-B4has the ability to act as a receptor for taste transduction, asdescribed in Example II.

GPCR-B4 encodes GPCRs with seven transmembrane regions that have“G-protein coupled receptor activity,” e.g., they bind to G-proteins inresponse to extracellular stimuli and promote production of secondmessengers such as IP3, cAMP, and Ca²⁺ via stimulation of enzymes suchas phospholipase C and adenylate cyclase (for a description of thestructure and function of GPCRs, see, e.g., Fong, supra, and Baldwin,supra).

The term GPCR-B4 therefore refers to polymorphic variants, alleles,mutants, and interspecies homologs that: (1) have about 70% amino acidsequence identity, optionally about 75, 80, 85, 90, or 95% amino acidsequence identity to SEQ ID NOS:1–2 and 7 over a window of about 25amino acids, optionally 50–100 amino acids; (2) bind to antibodiesraised against an immunogen comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 1–2 and 7 and conservativelymodified variants thereof; (3) specifically hybridize (with a size of atleast about 500, optionally at least about 900 nucleotides) understringent hybridization conditions to a sequence selected from the groupconsisting of SEQ ID NO:3–4 and 8, and conservatively modified variantsthereof; or (4) are amplified by primers that specifically hybridizeunder stringent hybridization conditions to the same sequence as adegenerate primer sets encoding SEQ ID NOS:5–6.

Topologically, sensory GPCRs have an N-terminal “extracellular domain,”a “transmembrane domain” comprising seven transmembrane regions andcorresponding cytoplasmic and extracellular loops, and a C-terminal“cytoplasmic domain” (see, e.g., Hoon et al., Cell 96:541–551 (1999);Buck & Axel, Cell 65:175–187 (1991)). These domains can be structurallyidentified using methods known to those of skill in the art, such assequence analysis programs that identify hydrophobic and hydrophilicdomains (see, e.g., Kyte & Doolittle, J. Mol. Biol. 157:105–132 (1982)).Such domains are useful for making chimeric proteins and for in vitroassays of the invention.

“Extracellular domain” therefore refers to the domain of GPCR-B4 thatprotrudes from the cellular membrane and binds to extracellular ligand.This region starts at the N-terminus and ends approximately at theconserved glutamic acid at amino acid position 563 plus or minusapproximately 20 amino acids. The region corresponding to amino acids1–580 of SEQ ID NO:1 (nucleotides 1–1740, with nucleotide 1 starting atthe ATG initiator methionine codon) is one embodiment of anextracellular domain that extends slightly into the transmembranedomain. This embodiment is useful for in vitro ligand binding assays,both soluble and solid phase.

“Transmembrane domain,” comprising seven transmembrane regions plus thecorresponding cytoplasmic and extracellular loops, refers to the domainof GPCR-B4 that starts approximately at the conserved glutamic acidresidue at amino acid position 563 plus or minus approximately 20 aminoacids and ends approximately at the conserved tyrosine amino acidresidue at position 812 plus or minus approximately 10 amino acids.

“Cytoplasmic domain” refers to the domain of GPCR-B4 that starts at theconserved tyrosine amino acid residue at position 812 plus or minusapproximately 10 amino acids and continues to the C-terminus of thepolypeptide.

“Biological sample” as used herein is a sample of biological tissue orfluid that contains GPCR-B4 or nucleic acid encoding GPCR-B4 protein.Such samples include, but are not limited to, tissue isolated fromhumans, mice, and rats, in particular, ton. Biological samples may alsoinclude sections of tissues such as frozen sections taken forhistological purposes. A biological sample is typically obtained from aeukaryotic organism, such as insects, protozoa, birds, fish, reptiles,and preferably a mammal such as rat, mouse, cow, dog, guinea pig, orrabbit, and most preferably a primate such as chimpanzees or humans.Tissues include tongue tissue, isolated taste buds, and testis tissue.

“GPCR activity” refers to the ability of a GPCR to transduce a signal.Such activity can be measured in a heterologous cell, by coupling a GPCR(or a chimeric GPCR) to either a G-protein or promiscuous G-protein suchas Gα15, and an enzyme such as PLC, and measuring increases inintracellular calcium using (Offermans & Simon, J. Biol. Chem.270:15175–15180 (1995)). Receptor activity can be effectively measuredby recording ligand-induced changes in [Ca²⁺]_(i) using fluorescentCa²⁺-indicator dyes and fluorometric imaging. Optionally, thepolypeptides of the invention are involved in sensory transduction,optionally taste transduction in taste cells.

The phrase “functional effects” in the context of assays for testingcompounds that modulate GPCR-B4 mediated taste transduction includes thedetermination of any parameter that is indirectly or directly under theinfluence of the receptor, e.g., functional, physical and chemicaleffects. It includes ligand binding, changes in ion flux, membranepotential, current flow, transcription, G-protein binding, GPCRphosphorylation or dephosphorylation, signal transduction,receptor-ligand interactions, second messenger concentrations (e.g.,cAMP, IP3, or intracellular Ca²⁺), in vitro, in vivo, and ex vivo andalso includes other physiologic effects such increases or decreases ofneurotransmitter or hormone release.

By “determining the functional effect” is meant assays for a compoundthat increases or decreases a parameter that is indirectly or directlyunder the influence of GPCR-B4, e.g., functional, physical and chemicaleffects. Such functional effects can be measured by any means known tothose skilled in the art, e.g., changes in spectroscopic characteristics(e.g., fluorescence, absorbance, refractive index), hydrodynamic (e.g.,shape), chromatographic, or solubility properties, patch clamping,voltage-sensitive dyes, whole cell currents, radioisotope efflux,inducible markers, oocyte GPCR-B4 expression; tissue culture cellGPCR-B4 expression; transcriptional activation of GPCR-B4; ligandbinding assays; voltage, membrane potential and conductance changes; ionflux assays; changes in intracellular second messengers such as cAMP andinositol triphosphate (IP3); changes in intracellular calcium levels;neurotransmitter release, and the like.

“Inhibitors,” “activators,” and “modulators” of GPCR-B4 are usedinterchangeably to refer to inhibitory, activating, or modulatingmolecules identified using in vitro and in vivo assays for tastetransduction, e.g., ligands, agonists, antagonists, and their homologsand mimetics. Inhibitors are compounds that, e.g., bind to, partially ortotally block stimulation, decrease, prevent, delay activation,inactivate, desensitize, or down regulate taste transduction, e.g.,antagonists. Activators are compounds that, e.g., bind to, stimulate,increase, open, activate, facilitate, enhance activation, sensitize orup regulate taste transduction, e.g., agonists. Modulators includecompounds that, e.g., alter the interaction of a receptor with:extracellular proteins that bind activators or inhibitor (e.g., ebnerinand other members of the hydrophobic carrier family); G-proteins;kinases (e.g., homologs of rhodopsin kinase and beta adrenergic receptorkinases that are involved in deactivation and desensitization of areceptor); and arrestin-like proteins, which also deactivate anddesensitize receptors. Modulators include genetically modified versionsof GPCR-B4, e.g., with altered activity, as well as naturally occurringand synthetic ligands, antagonists, agonists, small chemical moleculesand the like. Such assays for inhibitors and activators include, e.g.,expressing GPCR-B4 in cells or cell membranes, applying putativemodulator compounds, and then determining the functional effects ontaste transduction, as described above. Samples or assays comprisingGPCR-B4 that are treated with a potential activator, inhibitor, ormodulator are compared to control samples without the inhibitor,activator, or modulator to examine the extent of inhibition. Controlsamples (untreated with inhibitors) are assigned a relative GPCR-B4activity value of 100%. Inhibition of GPCR-B4 is achieved when theGPCR-B4 activity value relative to the control is about 80%, optionally50% or 25–0%. Activation of GPCR-B4 is achieved when the GPCR-B4activity value relative to the control is 110%, optionally 150%,optionally 200–500%, or 1000–3000% higher.

“Biologically active” GPCR-B4 refers to GPCR-B4 having GPCR activity asdescribed above, involved in taste transduction in taste receptor cells.

The terms “isolated” “purified” or “biologically pure” refer to materialthat is substantially or essentially free from components which normallyaccompany it as found in its native state. Purity and homogeneity aretypically determined using analytical chemistry techniques such aspolyacrylamide gel electrophoresis or high performance liquidchromatography. A protein that is the predominant species present in apreparation is substantially purified. In particular, an isolatedGPCR-B4 nucleic acid is separated from open reading frames that flankthe GPCR-B4 gene and encode proteins other than GPCR-B4. The term“purified” denotes that a nucleic acid or protein gives rise toessentially one band in an electrophoretic gel. Particularly, it meansthat the nucleic acid or protein is at least 85% pure, optionally atleast 95% pure, and optionally at least 99% pure.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form. The termencompasses nucleic acids containing known nucleotide analogs ormodified backbone residues or linkages, which are synthetic, naturallyoccurring, and non-naturally occurring, which have similar bindingproperties as the reference nucleic acid, and which are metabolized in amanner similar to the reference nucleotides. Examples of such analogsinclude, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides,peptide-nucleic acids (PNAs).

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605–2608(1985); Rossolini et al., Mol. Cell. Probes 8:91–98 (1994)). The termnucleic acid is used interchangeably with gene, cDNA, mRNA,oligonucleotide, and polynucleotide.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an α carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another:

-   1) Alanine (A), Glycine (G);-   2) Aspartic acid (D), Glutamic acid (E);-   3) Asparagine (N), Glutamine (Q);-   4) Arginine (R), Lysine (K);-   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);-   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);-   7) Serine (S), Threonine (T); and-   8) Cysteine (C), Methionine (M)-   (see, e.g., Creighton, Proteins (1984)).

Macromolecular structures such as polypeptide structures can bedescribed in terms of various levels of organization. For a generaldiscussion of this organization, see, e.g., Alberts et al., MolecularBiology of the Cell (3^(rd ed.,) 1994) and Cantor and Schimmel,Biophysical Chemistry Part I: The Conformation of BiologicalMacromolecules (1980). “Primary structure” refers to the amino acidsequence of a particular peptide. “Secondary structure” refers tolocally ordered, three dimensional structures within a polypeptide.These structures are commonly known as domains. Domains are portions ofa polypeptide that form a compact unit of the polypeptide and aretypically 50 to 350 amino acids long. Typical domains are made up ofsections of lesser organization such as stretches of β-sheet andα-helices. “Tertiary structure” refers to the complete three dimensionalstructure of a polypeptide monomer. “Quaternary structure” refers to thethree dimensional structure formed by the noncovalent association ofindependent tertiary units. Anisotropic terms are also known as energyterms.

A “label” or a “detectable moiety” is a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, or chemicalmeans. For example, useful labels include ³²P, fluorescent dyes,electron-dense reagents, enzymes (e.g., as commonly used in an ELISA),biotin, digoxigenin, or haptens and proteins for which ant or 7 can bemade detectable, e.g., by incorporating a radiolabel into the peptide,and used to detect antibodies specifically reactive with the peptide).

A “labeled nucleic acid probe or oligonucleotide” is one that is bound,either covalently, through a linker or a chemical bond, ornoncovalently, through ionic, van der Waals, electrostatic, or hydrogenbonds to a label such that the presence of the probe may be detected bydetecting the presence of the label bound to the probe.

As used herein a “nucleic acid probe or oligonucleotide” is defined as anucleic acid capable of binding to a target nucleic acid ofcomplementary sequence through one or more types of chemical bonds,usually through complementary base pairing, usually through hydrogenbond formation. As used herein, a probe may include natural (i.e., A, G,C, or T) or modified bases (7-deazaguanosine, inosine, etc.). Inaddition, the bases in a probe may be joined by a linkage other than aphosphodiester bond, so long as it does not interfere withhybridization. Thus, for example, probes may be peptide nucleic acids inwhich the constituent bases are joined by peptide bonds rather thanphosphodiester linkages. It will be understood by one of skill in theart that probes may bind target sequences lacking completecomplementarity with the probe sequence depending upon the stringency ofthe hybridization conditions. The probes are optionally directly labeledas with isotopes, chromophores, lumiphores, chromogens, or indirectlylabeled such as with biotin to which a streptavidin complex may laterbind. By assaying for the presence or absence of the probe, one candetect the presence or absence of the select sequence or subsequence.

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all.

The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not found in the same relationship to each otherin nature. For instance, the nucleic acid is typically recombinantlyproduced, having two or more sequences from unrelated genes arranged tomake a new functional nucleic acid, e.g., a promoter from one source anda coding region from another source. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature (e.g., afusion protein).

A “promoter” is defined as an array of nucleic acid control sequencesthat direct transcription of a nucleic acid. As used herein, a promoterincludes necessary nucleic acid sequences near the start site oftranscription, such as, in the case of a polymerase II type promoter, aTATA element. A promoter also optionally includes distal enhancer orrepressor elements, which can be located as much as several thousandbase pairs from the start site of transcription. A “constitutive”promoter is a promoter that is active under most environmental anddevelopmental conditions. An “inducible” promoter is a promoter that isactive under environmental or developmental regulation. The term“operably linked” refers to a functional linkage between a nucleic acidexpression control sequence (such as a promoter, or array oftranscription factor binding sites) and a second nucleic acid sequence,wherein the expression control sequence directs transcription of thenucleic acid corresponding to the second sequence.

An “expression vector” is a nucleic acid construct, generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in ahost cell. The expression vector can be part of a plasmid, virus, ornucleic acid fragment. Typically, the expression vector includes anucleic acid to be transcribed operably linked to a promoter.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., 70% identity, optionally 75%, 80%, 85%, 90%, or 95% identity overa specified region), when compared and aligned for maximumcorrespondence over a comparison window, or designated region asmeasured using one of the following sequence comparison algorithms or bymanual alignment and visual inspection. Such sequences are then said tobe “substantially identical.” This definition also refers to thecompliment of a test sequence. Optionally, the identity exists over aregion that is at least about 50 amino acids or nucleotides in length,or more preferably over a region that is 75–100 amino acids ornucleotides in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),by the search for similarity method of Pearson & Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., CurrentProtocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

One example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments to show relationship and percent sequence identity.It also plots a tree or dendogram showing the clustering relationshipsused to create the alignment. PILEUP uses a simplification of theprogressive alignment method of Feng & Doolittle, J. Mol. Evol.35:351–360 (1987). The method used is similar to the method described byHiggins & Sharp, CABIOS 5:151–153 (1989). The program can align up to300 sequences, each of a maximum length of 5,000 nucleotides or aminoacids. The multiple alignment procedure begins with the pairwisealignment of the two most similar sequences, producing a cluster of twoaligned sequences. This cluster is then aligned to the next most relatedsequence or cluster of aligned sequences. Two clusters of sequences arealigned by a simple extension of the pairwise alignment of twoindividual sequences. The final alignment is achieved by a series ofprogressive, pairwise alignments. The program is run by designatingspecific sequences and their amino acid or nucleotide coordinates forregions of sequence comparison and by designating the programparameters. Using PILEUP, a reference sequence is compared to other testsequences to determine the percent sequence identity relationship usingthe following parameters: default gap weight (3.00), default gap lengthweight (0.10), and weighted end gaps. PILEUP can be obtained from theGCG sequence analysis software package, e.g., version 7.0 (Devereaux etal., Nuc. Acids Res. 12:387–395 (1984).

Another example of algorithm that is suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al., Nuc. Acids Res.25:3389–3402 (1977) and Altschul et al., J. Mol. Biol. 215:403–410(1990), respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al., supra). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) or 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989))alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin & Altschul, Proc.Nat'l. Acad. Sci. USA 90:5873–5787 (1993)). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

An indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the antibodiesraised against the polypeptide encoded by the second nucleic acid, asdescribed below. Thus, a polypeptide is typically substantiallyidentical to a second polypeptide, for example, where the two peptidesdiffer only by conservative substitutions. Another indication that twonucleic acid sequences are substantially identical is that the twomolecules or their complements hybridize to each other under stringentconditions, as described below. Yet another indication that two nucleicacid sequences are substantially identical is that the same primers canbe used to amplify the sequence.

The phrase “selectively (or specifically) hybridizes to” refers to thebinding, duplexing, or hybridizing of a molecule only to a particularnucleotide sequence under stringent hybridization conditions when thatsequence is present in a complex mixture (e.g., total cellular orlibrary DNA or RNA).

The phrase “stringent hybridization conditions” refers to conditionsunder which a probe will hybridize to its target subsequence, typicallyin a complex mixture of nucleic acid, but to no other sequences.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures. An extensive guide to the hybridization of nucleicacids is found in Tijssen, Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Probes, “Overview of principles ofhybridization and the strategy of nucleic acid assays” (1993).Generally, stringent conditions are selected to be about 5–10° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength pH. The T_(m) is the temperature (under definedionic strength, pH, and nucleic concentration) at which 50% of theprobes complementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at T_(m),50% of the probes are occupied at equilibrium). Stringent conditionswill be those in which the salt concentration is less than about 1.0 Msodium ion, typically about 0.01 to 1.0 M sodium ion concentration (orother salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C. for long probes (e.g., greater than 50 nucleotides). Stringentconditions may also be achieved with the addition of destabilizingagents such as formamide. For selective or specific hybridization, apositive signal is at least two times background, optionally 10 timesbackground hybridization. Exemplary stringent hybridization conditionscan be as following: 50% formamide, 5×SSC, and 1% SDS, incubating at 42°C., or, 5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and0.1% SDS at 65° C.

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. A positive hybridization is at least twicebackground. Those of ordinary skill will readily recognize thatalternative hybridization and wash conditions can be utilized to provideconditions of similar stringency.

“Antibody” refers to a polypeptide comprising a framework region from animmunoglobulin gene or fragments thereof that specifically binds andrecognizes an antigen. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon, and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

An exemplary immunoglobulin (antibody) structural unit comprises atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” chain (about 50–70 kDa). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

Antibodies exist, e.g., as intact immunoglobulins or as a number ofwell-characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)′₂, a dimer ofFab which itself is a light chain joined to V_(H)—C_(H)1 by a disulfidebond. The F(ab)′₂ may be reduced under mild conditions to break thedisulfide linkage in the hinge region, thereby converting the F(ab)′₂dimer into an Fab′ monomer. The Fab′ monomer is essentially Fab withpart of the hinge region (see Fundamental Immunology (Paul ed., 3d ed.1993). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchfragments may be synthesized de novo either chemically or by usingrecombinant DNA methodology. Thus, the term antibody, as used herein,also includes antibody fragments either produced by the modification ofwhole antibodies, or those synthesized de novo using recombinant DNAmethodologies (e.g., single chain Fv) or those identified using phagedisplay libraries (see, e.g., McCafferty et al., Nature 348:552–554(1990)).

For preparation of monoclonal or polyclonal antibodies, any techniqueknown in the art can be used (see, e.g., Kohler & Milstein, Nature256:495–497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Coleet al., pp. 77–96 in Monoclonal Antibodies and Cancer Therapy (1985)).Techniques for the production of single chain antibodies (U.S. Pat. No.4,946,778) can be adapted to produce antibodies to polypeptides of thisinvention. Also, transgenic mice, or other organisms such as othermammals, may be used to express humanized antibodies. Alternatively,phage display technology can be used to identify antibodies andheteromeric Fab fragments that specifically bind to selected antigens(see, e.g., McCafferty et al., Nature 348:552–554 (1990); Marks et al.,Biotechnology 10:779–783 (1992)).

A “chimeric antibody” is an antibody molecule in which (a) the constantregion, or a portion thereof, is altered, replaced or exchanged so thatthe antigen binding site (variable region) is linked to a constantregion of a different or altered class, effector function and/orspecies, or an entirely different molecule which confers new propertiesto the chimeric antibody, e.g., an enzyme, toxin, hormone, growthfactor, drug, etc.; or (b) the variable region, or a portion thereof, isaltered, replaced or exchanged with a variable region having a differentor altered antigen specificity.

An “anti-GPCR-B4” antibody is an antibody or antibody fragment thatspecifically binds a polypeptide encoded by the GPCR-B4 gene, cDNA, or asubsequence thereof.

The term “immunoassay” is an assay that uses an antibody to specificallybind an antigen. The immunoassay is characterized by the use of specificbinding properties of a particular antibody to isolate, target, and/orquantify the antigen.

The phrase “specifically (or selectively) binds” to an antibody or“specifically (or selectively) immunoreactive with,” when referring to aprotein or peptide, refers to a binding reaction that is determinativeof the presence of the protein in a heterogeneous population of proteinsand other biologics. Thus, under designated immunoassay conditions, thespecified antibodies bind to a particular protein at least two times thebackground and do not substantially bind in a significant amount toother proteins present in the sample. Specific binding to an antibodyunder such conditions may require an antibody that is selected for itsspecificity for a particular protein. For example, polyclonal antibodiesraised to GPCR-B4 from specific species such as rat, mouse, or human canbe selected to obtain only those polyclonal antibodies that arespecifically immunoreactive with GPCR-B4 and not with other proteins,except for polymorphic variants and alleles of GPCR-B4. This selectionmay be achieved by subtracting out antibodies that cross-react withGPCR-B4 molecules from other species. A variety of immunoassay formatsmay be used to select antibodies specifically immunoreactive with aparticular protein. For example, solid-phase ELISA immunoassays areroutinely used to select antibodies specifically immunoreactive with aprotein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual(1988), for a description of immunoassay formats and conditions that canbe used to determine specific immunoreactivity). Typically a specific orselective reaction will be at least twice background signal or noise andmore typically more than 10 to 100 times background.

The phrase “selectively associates with” refers to the ability of anucleic acid to “selectively hybridize” with another as defined above,or the ability of an antibody to “selectively (or specifically) bind toa protein, as defined above.

By “host cell” is meant a cell that contains an expression vector andsupports the replication or expression of the expression vector. Hostcells may be prokaryotic cells such as E. coli, or eukaryotic cells suchas yeast, insect, amphibian, or mammalian cells such as CHO, HeLa andthe like, e.g., cultured cells, explants, and cells in vivo.

III. Isolation of the Nucleic Acid Encoding GPCR-B4

A. General recombinant DNA methods

This invention relies on routine techniques in the field of recombinantgenetics. Basic texts disclosing the general methods of use in thisinvention include Sambrook et al., Molecular Cloning, A LaboratoryManual (2nd ed. 1989); Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990); and Current Protocols in Molecular Biology(Ausubel et al., eds., 1994)).

For nucleic acids, sizes are given in either kilobases (kb) or basepairs (bp). These are estimates derived from agarose or acrylamide gelelectrophoresis, from sequenced nucleic acids, or from published DNAsequences. For proteins, sizes are given in kilodaltons (kDa) or aminoacid residue numbers. Proteins sizes are estimated from gelelectrophoresis, from sequenced proteins, from derived amino acidsequences, or from published protein sequences.

Oligonucleotides that are not commercially available can be chemicallysynthesized according to the solid phase phosphoramidite triester methodfirst described by Beaucage & Caruthers, Tetrahedron Letts. 22:1859–1862(1981), using an automated synthesizer, as described in Van Devanter et.al., Nucleic Acids Res. 12:6159–6168 (1984). Purification ofoligonucleotides is by either native acrylamide gel electrophoresis orby anion-exchange HPLC as described in Pearson & Reanier, J. Chrom.255:137–149 (1983).

The sequence of the cloned genes and synthetic oligonucleotides can beverified after cloning using, e.g., the chain termination method forsequencing double-stranded templates of Wallace et al., Gene 16:21–26(1981).

B. Cloning Methods for the Isolation of Nucleotide Sequences EncodingGPCR-B4

In general, the nucleic acid sequences encoding GPCR-B4 and relatednucleic acid sequence homologs are cloned from cDNA and genomic DNAlibraries by hybridization with a probe, or isolated using amplificationtechniques with oligonucleotide primers. For example, GPCR-B4 sequencesare typically isolated from mammalian nucleic acid (genomic or cDNA)libraries by hybridizing with a nucleic acid probe, the sequence ofwhich can be derived from SEQ ID NOS:3–4 and 8. A suitable tissue fromwhich GPCR-B4 RNA and cDNA can be isolated is tongue tissue, optionallytaste bud tissues or individual taste cells.

Amplification techniques using primers can also be used to amplify andisolate GPCR-B4 from DNA or RNA. The degenerate primers encoding thefollowing amino acid sequences can also be used to amplify a sequence ofGPCR-B4: SEQ ID NOS:5–6 (see, e.g., Dieffenfach & Dveksler, PCR Primer:A Laboratory Manual (1995)). These primers can be used, e.g., to amplifyeither the full length sequence or a probe of one to several hundrednucleotides, which is then used to screen a mammalian library forfull-length GPCR-B4.

Nucleic acids encoding GPCR-B4 can also be isolated from expressionlibraries using antibodies as probes. Such polyclonal or monoclonalantibodies can be raised using the sequence of SEQ ID NOS:1–2 and 7.

GPCR-B4 polymorphic variants, alleles, and interspecies homologs thatare substantially identical to GPCR-B4 can be isolated using GPCR-B4nucleic acid probes, and oligonucleotides under stringent hybridizationconditions, by screening libraries. Alternatively, expression librariescan be used to clone GPCR-B4 and GPCR-B4 polymorphic variants, alleles,and interspecies homologs, by detecting expressed homologsimmunologically with antisera or purified antibodies made againstGPCR-B4, which also recognize and selectively bind to the GPCR-B4homolog.

To make a cDNA library, one should choose a source that is rich inGPCR-B4 mRNA, e.g., tongue tissue, or isolated taste buds. The mRNA isthen made into cDNA using reverse transcriptase, ligated into arecombinant vector, and transfected into a recombinant host forpropagation, screening and cloning. Methods for making and screeningcDNA libraries are well known (see, e.g., Gubler & Hoffman, Gene25:263–269 (1983); Sambrook et al., supra; Ausubel et al., supra).

For a genomic library, the DNA is extracted from the tissue and eithermechanically sheared or enzymatically digested to yield fragments ofabout 12–20 kb. The fragments are then separated by gradientcentrifugation from undesired sizes and are constructed in bacteriophagelambda vectors. These vectors and phage are packaged in vitro.Recombinant phage are analyzed by plaque hybridization as described inBenton & Davis, Science 196:180–182 (1977). Colony hybridization iscarried out as generally described in Grunstein et al., Proc. Natl.Acad. Sci. USA., 72:3961–3965 (1975).

An alternative method of isolating GPCR-B4 nucleic acid and its homologscombines the use of synthetic oligonucleotide primers and amplificationof an RNA or DNA template (see U.S. Pat. Nos. 4,683,195 and 4,683,202;PCR Protocols: A Guide to Methods and Applications (Innis et al., eds,1990)). Methods such as polymerase chain reaction (PCR) and ligase chainreaction (LCR) can be used to amplify nucleic acid sequences of GPCR-B4directly from mRNA, from cDNA, from genomic libraries or cDNA libraries.Degenerate oligonucleotides can be designed to amplify GPCR-B4 homologsusing the sequences provided herein. Restriction endonuclease sites canbe incorporated into the primers. Polymerase chain reaction or other invitro amplification methods may also be useful, for example, to clonenucleic acid sequences that code for proteins to be expressed, to makenucleic acids to use as probes for detecting the presence of GPCR-B4encoding mRNA in physiological samples, for nucleic acid sequencing, orfor other purposes. Genes amplified by the PCR reaction can be purifiedfrom agarose gels and cloned into an appropriate vector.

Gene expression of GPCR-B4 can also be analyzed by techniques known inthe art, e.g., reverse transcription and amplification of mRNA,isolation of total RNA or poly A⁺ RNA, northern blotting, dot blotting,in situ hybridization, RNase protection, probing DNA microchip arrays,and the like. In one embodiment, high density oligonucleotide analysistechnology (e.g., GeneChip™) is used to identify homologs andpolymorphic variants of the GPCRs of the invention. In the case wherethe homologs being identified are linked to a known disease, they can beused with GeneChip™ as a diagnostic tool in detecting the disease in abiological sample, see, e.g., Gunthand et al., AIDS Res. Hum.Retroviruses 14: 869–876 (1998); Kozal et al., Nat. Med. 2:753–759(1996); Matson et al., Anal. Biochem. 224:110–106 (1995); Lockhart etal., Nat. Biotechnol. 14:1675–1680 (1996); Gingeras et al., Genome Res.8:435–448 (1998); Hacia et al., Nucleic Acids Res. 26:3865–3866 (1998).

Synthetic oligonucleotides can be used to construct recombinant GPCR-B4genes for use as probes or for expression of protein. This method isperformed using a series of overlapping oligonucleotides usually 40–120bp in length, representing both the sense and nonsense strands of thegene. These DNA fragments are then annealed, ligated and cloned.Alternatively, amplification techniques can be used with precise primersto amplify a specific subsequence of the GPCR-B4 nucleic acid. Thespecific subsequence is then ligated into an expression vector.

The nucleic acid encoding GPCR-B4 is typically cloned into intermediatevectors before transformation into prokaryotic or eukaryotic cells forreplication and/or expression. These intermediate vectors are typicallyprokaryote vectors, e.g., plasmids, or shuttle vectors.

Optionally, nucleic acids encoding chimeric proteins comprising GPCR-B4or domains thereof can be made according to standard techniques. Forexample, a domain such as ligand binding domain, an extracellulardomain, a transmembrane domain (e.g., one comprising seven transmembraneregions and corresponding extracellular and cytosolic loops), thetransmembrane domain and a cytoplasmic domain, an active site, a subunitassociation region, etc., can be covalently linked to a heterologousprotein. For example, an extracellular domain can be linked to aheterologous GPCR transmembrane domain, or a heterologous GPCRextracellular domain can be linked to a transmembrane domain. Otherheterologous proteins of choice include, e.g., green fluorescentprotein, β-gal, glutamate receptor, and the rhodopsin presequence.

C. Expression in Prokaryotes and Eukaryotes

To obtain high level expression of a cloned gene or nucleic acid, suchas those cDNAs encoding GPCR-B4, one typically subclones GPCR-B4 into anexpression vector that contains a strong promoter to directtranscription, a transcription/translation terminator, and if for anucleic acid encoding a protein, a ribosome binding site fortranslational initiation. Suitable bacterial promoters are well known inthe art and described, e.g., in Sambrook et al. and Ausubel et al.Bacterial expression systems for expressing the GPCR-B4 protein areavailable in, e.g., E. coli, Bacillus sp., and Salmonella (Palva et al.,Gene 22:229–235 (1983); Mosbach et al., Nature 302:543–545 (1983). Kitsfor such expression systems are commercially available. Eukaryoticexpression systems for mammalian cells, yeast, and insect cells are wellknown in the art and are also commercially available. In one embodiment,the eukaryotic expression vector is an adenoviral vector, anadeno-associated vector, or a retroviral vector.

The promoter used to direct expression of a heterologous nucleic aciddepends on the particular application. The promoter is optionallypositioned about the same distance from the heterologous transcriptionstart site as it is from the transcription start site in its naturalsetting. As is known in the art, however, some variation in thisdistance can be accommodated without loss of promoter function.

In addition to the promoter, the expression vector typically contains atranscription unit or expression cassette that contains all theadditional elements required for the expression of the GPCR-B4 encodingnucleic acid in host cells. A typical expression cassette thus containsa promoter operably linked to the nucleic acid sequence encoding GPCR-B4and signals required for efficient polyadenylation of the transcript,ribosome binding sites, and translation termination. The nucleic acidsequence encoding GPCR-B4 may typically be linked to a cleavable signalpeptide sequence to promote secretion of the encoded protein by thetransformed cell. Such signal peptides would include, among others, thesignal peptides from tissue plasminogen activator, insulin, and neurongrowth factor, and juvenile hormone esterase of Heliothis virescens.Additional elements of the cassette may include enhancers and, ifgenomic DNA is used as the structural gene, introns with functionalsplice donor and acceptor sites.

In addition to a promoter sequence, the expression cassette should alsocontain a transcription termination region downstream of the structuralgene to provide for efficient termination. The termination region may beobtained from the same gene as the promoter sequence or may be obtainedfrom different genes.

The particular expression vector used to transport the geneticinformation into the cell is not particularly critical. Any of theconventional vectors used for expression in eukaryotic or prokaryoticcells may be used. Standard bacterial expression vectors includeplasmids such as pBR322 based plasmids, pSKF, pET23D, and fusionexpression systems such as GST and LacZ. Epitope tags can also be addedto recombinant proteins to provide convenient methods of isolation,e.g., c-myc.

Expression vectors containing regulatory elements from eukaryoticviruses are typically used in eukaryotic expression vectors, e.g., SV40vectors, papilloma virus vectors, and vectors derived from Epstein-Barrvirus. Other exemplary eukaryotic vectors include pMSG, pAV009/A⁺,pMTO10/A⁺, pMAMneo-5, baculovirus pDSVE, and any other vector allowingexpression of proteins under the direction of the SV40 early promoter,SV40 later promoter, metallothionein promoter, murine mammary tumorvirus promoter, Rous sarcoma virus promoter, polyhedrin promoter, orother promoters shown effective for expression in eukaryotic cells.

Some expression systems have markers that provide gene amplificationsuch as thymidine kinase, hygromycin B phosphotransferase, anddihydrofolate reductase. Alternatively, high yield expression systemsnot involving gene amplification are also suitable, such as using abaculovirus vector in insect cells, with a GPCR-B4 encoding sequenceunder the direction of the polyhedrin promoter or other strongbaculovirus promoters.

The elements that are typically included in expression vectors alsoinclude a replicon that functions in E. coli, a gene encoding antibioticresistance to permit selection of bacteria that harbor recombinantplasmids, and unique restriction sites in nonessential regions of theplasmid to allow insertion of eukaryotic sequences. The particularantibiotic resistance gene chosen is not critical, any of the manyresistance genes known in the art are suitable. The prokaryoticsequences are optionally chosen such that they do not interfere with thereplication of the DNA in eukaryotic cells, if necessary.

Standard transfection methods are used to produce bacterial, mammalian,yeast or insect cell lines that express large quantities of GPCR-B4protein, which are then purified using standard techniques (see, e.g.,Colley et al., J. Biol. Chem. 264:17619–17622 (1989); Guide to ProteinPurification, in Methods in Enzymology, vol. 182 (Deutscher, ed.,1990)). Transformation of eukaryotic and prokaryotic cells are performedaccording to standard techniques (see, e.g., Morrison, J. Bact.132:349–351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology101:347–362 (Wu et al., eds, 1983).

Any of the well known procedures for introducing foreign nucleotidesequences into host cells may be used. These include the use of calciumphosphate transfection, polybrene, protoplast fusion, electroporation,liposomes, microinjection, plasma vectors, viral vectors and any of theother well known methods for introducing cloned genomic DNA, cDNA,synthetic DNA or other foreign genetic material into a host cell (see,e.g., Sambrook et al., supra). It is only necessary that the particulargenetic engineering procedure used be capable of successfullyintroducing at least one gene into the host cell capable of expressingGPCR-B4.

After the expression vector is introduced into the cells, thetransfected cells are cultured under conditions favoring expression ofGPCR-B4, which is recovered from the culture using standard techniquesidentified below.

IV. Purification of GPCR-B4

Either naturally occurring or recombinant GPCR-B4 can be purified foruse in functional assays. Optionally, recombinant GPCR-B4 is purified.Naturally occurring GPCR-B4 is purified, e.g., from mammalian tissuesuch as tongue tissue, and any other source of a GPCR-B4 homolog.Recombinant GPCR-B4 is purified from any suitable bacterial oreukaryotic expression system, e.g., CHO cells or insect cells.

GPCR-B4 may be purified to substantial purity by standard techniques,including selective precipitation with such substances as ammoniumsulfate; column chromatography, immunopurification methods, and others(see, e.g., Scopes, Protein Purification: Principles and Practice(1982); U.S. Pat. No. 4,673,641; Ausubel et al., supra; and Sambrook etal., supra).

A number of procedures can be employed when recombinant GPCR-B4 is beingpurified. For example, proteins having established molecular adhesionproperties can be reversible fused to GPCR-B4. With the appropriateligand, GPCR-B4 can be selectively adsorbed to a purification column andthen freed from the column in a relatively pure form. The fused proteinis then removed by enzymatic activity. Finally GPCR-B4 could be purifiedusing immunoaffinity columns.

A. Purification of GPCR-B4from Recombinant Cells

Recombinant proteins are expressed by transformed bacteria or eukaryoticcells such as CHO cells or insect cells in large amounts, typicallyafter promoter induction; but expression can be constitutive. Promoterinduction with IPTG is a one example of an inducible promoter system.Cells are grown according to standard procedures in the art. Fresh orfrozen cells are used for isolation of protein.

Proteins expressed in bacteria may form insoluble aggregates (“inclusionbodies”). Several protocols are suitable for purification of GPCR-B4inclusion bodies. For example, purification of inclusion bodiestypically involves the extraction, separation and/or purification ofinclusion bodies by disruption of bacterial cells, e.g., by incubationin a buffer of 50 mM TRIS/HCL pH 7.5, 50 mM NaCl, 5 mM MgCl₂, 1 mM DTT,0.1 mM ATP, and 1 mM PMSF. The cell suspension can be lysed using 2–3passages through a French Press, homogenized using a Polytron (BrinkmanInstruments) or sonicated on ice. Alternate methods of lysing bacteriaare apparent to those of skill in the art (see, e.g., Sambrook et al.,supra; Ausubel et al., supra).

If necessary, the inclusion bodies are solubilized, and the lysed cellsuspension is typically centrifuged to remove unwanted insoluble matter.Proteins that formed the inclusion bodies may be renatured by dilutionor dialysis with a compatible buffer. Suitable solvents include, but arenot limited to urea (from about 4 M to about 8 M), formamide (at leastabout 80%, volume/volume basis), and guanidine hydrochloride (from about4 M to about 8 M). Some solvents which are capable of solubilizingaggregate-forming proteins, for example SDS (sodium dodecyl sulfate),70% formic acid, are inappropriate for use in this procedure due to thepossibility of irreversible denaturation of the proteins, accompanied bya lack of immunogenicity and/or activity. Although guanidinehydrochloride and similar agents are denaturants, this denaturation isnot irreversible and renaturation may occur upon removal (by dialysis,for example) or dilution of the denaturant, allowing re-formation ofimmunologically and/or biologically active protein. Other suitablebuffers are known to those skilled in the art. GPCR-B4 is separated fromother bacterial proteins by standard separation techniques, e.g., withNi-NTA agarose resin.

Alternatively, it is possible to purify GPCR-B4 from bacteria periplasm.After lysis of the bacteria, when GPCR-B4 is exported into the periplasmof the bacteria, the periplasmic fraction of the bacteria can beisolated by cold osmotic shock in addition to other methods known toskill in the art. To isolate recombinant proteins from the periplasm,the bacterial cells are centrifuged to form a pellet. The pellet isresuspended in a buffer containing 20% sucrose. To lyse the cells, thebacteria are centrifuged and the pellet is resuspended in ice-cold 5 mMMgSO₄ and kept in an ice bath for approximately 10 minutes. The cellsuspension is centrifuged and the supernatant decanted and saved. Therecombinant proteins present in the supernatant can be separated fromthe host proteins by standard separation techniques well known to thoseof skill in the art.

B. Standard Protein Separation Techniques for Purifying GPCR-B4

Solubility Fractionation

Often as an initial step, particularly if the protein mixture iscomplex, an initial salt fractionation can separate many of the unwantedhost cell proteins (or proteins derived from the cell culture media)from the recombinant protein of interest. The preferred salt is ammoniumsulfate. Ammonium sulfate precipitates proteins by effectively reducingthe amount of water in the protein mixture. Proteins then precipitate onthe basis of their solubility. The more hydrophobic a protein is, themore likely it is to precipitate at lower ammonium sulfateconcentrations. A typical protocol includes adding saturated ammoniumsulfate to a protein solution so that the resultant ammonium sulfateconcentration is between 20–30%. This concentration will precipitate themost hydrophobic of proteins. The precipitate is then discarded (unlessthe protein of interest is hydrophobic) and ammonium sulfate is added tothe supernatant to a concentration known to precipitate the protein ofinterest. The precipitate is then solubilized in buffer and the excesssalt removed if necessary, either through dialysis or diafiltration.Other methods that rely on solubility of proteins, such as cold ethanolprecipitation, are well known to those of skill in the art and can beused to fractionate complex protein mixtures.

Size Differential Filtration

The molecular weight of GPCR-B4 can be used to isolated it from proteinsof greater and lesser size using ultrafiltration through membranes ofdifferent pore size (for example, Amicon or Millipore membranes). As afirst step, the protein mixture is ultrafiltered through a membrane witha pore size that has a lower molecular weight cut-off than the molecularweight of the protein of interest. The retentate of the ultrafiltrationis then ultrafiltered against a membrane with a molecular cut offgreater than the molecular weight of the protein of interest. Therecombinant protein will pass through the membrane into the filtrate.The filtrate can then be chromatographed as described below.

Column Chromatography

GPCR-B4 can also be separated from other proteins on the basis of itssize, net surface charge, hydrophobicity, and affinity for ligands. Inaddition, antibodies raised against proteins can be conjugated to columnmatrices and the proteins immunopurified. All of these methods are wellknown in the art. It will be apparent to one of skill thatchromatographic techniques can be performed at any scale and usingequipment from many different manufacturers (e.g., Pharmacia Biotech).

V. Immunological Detection of GPCR-B4

In addition to the detection of GPCR-B4 genes and gene expression usingnucleic acid hybridization technology, one can also use immunoassays todetect GPCR-B4, e.g., to identify taste receptor cells and variants ofGPCR-B4. Immunoassays can be used to qualitatively or quantitativelyanalyze GPCR-B4. A general overview of the applicable technology can befound in Harlow & Lane, Antibodies: A Laboratory Manual (1988).

A. Antibodies to GPCR-B4

Methods of producing polyclonal and monoclonal antibodies that reactspecifically with GPCR-B4 are known to those of skill in the art (see,e.g., Coligan, Current Protocols in Immunology (1991); Harlow & Lane,supra; Goding, Monoclonal Antibodies: Principles and Practice (2d ed.1986); and Kohler & Milstein, Nature 256:495–497 (1975). Such techniquesinclude antibody preparation by selection of antibodies from librariesof recombinant antibodies in phage or similar vectors, as well aspreparation of polyclonal and monoclonal antibodies by immunizingrabbits or mice (see, e.g., Huse et al., Science 246:1275–1281 (1989);Ward et al., Nature 341:544–546 (1989)).

A number of GPCR-B4 comprising immunogens may be used to produceantibodies specifically reactive with GPCR-B4. For example, recombinantGPCR-B4 or an antigenic fragment thereof, is isolated as describedherein. Recombinant protein can be expressed in eukaryotic orprokaryotic cells as described above, and purified as generallydescribed above. Recombinant protein is the preferred immunogen for theproduction of monoclonal or polyclonal antibodies. Alternatively, asynthetic peptide derived from the sequences disclosed herein andconjugated to a carrier protein can be used an immunogen. Naturallyoccurring protein may also be used either in pure or impure form. Theproduct is then injected into an animal capable of producing antibodies.Either monoclonal or polyclonal antibodies may be generated, forsubsequent use in immunoassays to measure the protein.

Methods of production of polyclonal antibodies are known to those ofskill in the art. An inbred strain of mice (e.g., BALB/C mice) orrabbits is immunized with the protein using a standard adjuvant, such asFreund's adjuvant, and a standard immunization protocol. The animal'simmune response to the immunogen preparation is monitored by taking testbleeds and determining the titer of reactivity to GPCR-B4. Whenappropriately high titers of antibody to the immunogen are obtained,blood is collected from the animal and antisera are prepared. Furtherfractionation of the antisera to enrich for antibodies reactive to theprotein can be done if desired (see Harlow & Lane, supra).

Monoclonal antibodies may be obtained by various techniques familiar tothose skilled in the art. Briefly, spleen cells from an animal immunizedwith a desired antigen are immortalized, commonly by fusion with amyeloma cell (see Kohler & Milstein, Eur. J. Immunol. 6:511–519 (1976)).Alternative methods of immortalization include transformation withEpstein Barr Virus, oncogenes, or retroviruses, or other methods wellknown in the art. Colonies arising from single immortalized cells arescreened for production of antibodies of the desired specificity andaffinity for the antigen, and yield of the monoclonal antibodiesproduced by such cells may be enhanced by various techniques, includinginjection into the peritoneal cavity of a vertebrate host.Alternatively, one may isolate DNA sequences which encode a monoclonalantibody or a binding fragment thereof by screening a DNA library fromhuman B cells according to the general protocol outlined by Huse et al.,Science 246:1275–1281 (1989).

Monoclonal antibodies and polyclonal sera are collected and titeredagainst the immunogen protein in an immunoassay, for example, a solidphase immunoassay with the immunogen immobilized on a solid support.Typically, polyclonal antisera with a titer of 10⁴ or greater areselected and tested for their cross reactivity against non-GPCR-B4proteins or even other related proteins from other organisms, using acompetitive binding immunoassay. Specific polyclonal antisera andmonoclonal antibodies will usually bind with a K_(d) of at least about0.1 mM, more usually at least about 1 μM, optionally at least about 0.1μM or better, and optionally 0.01 μM or better.

Once GPCR-B4 specific antibodies are available, GPCR-B4 can be detectedby a variety of immunoassay methods. For a review of immunological andimmunoassay procedures, see Basic and Clinical Immunology (Stites & Terreds., 7th ed. 1991). Moreover, the immunoassays of the present inventioncan be performed in any of several configurations, which are reviewedextensively in Enzyme Immunoassay (Maggio, ed., 1980); and Harlow &Lane, supra.

B. Immunological Binding Assays

GPCR-B4 can be detected and/or quantified using any of a number of wellrecognized immunological binding assays (see, e.g., U.S. Pat. Nos.4,366,241; 4,376,110; 4,517,288; and 4,837,168). For a review of thegeneral immunoassays, see also Methods in Cell Biology: Antibodies inCell Biology, volume 37 (Asai, ed. 1993); Basic and Clinical Immunology(Stites & Terr, eds., 7th ed. 1991). Immunological binding assays (orimmunoassays) typically use an antibody that specifically binds to aprotein or antigen of choice (in this case the GPCR-B4 or antigenicsubsequence thereof). The antibody (e.g., anti-GPCR-B4) may be producedby any of a number of means well known to those of skill in the art andas described above.

Immunoassays also often use a labeling agent to specifically bind to andlabel the complex formed by the antibody and antigen. The labeling agentmay itself be one of the moieties comprising the antibody/antigencomplex. Thus, the labeling agent may be a labeled GPCR-B4 polypeptideor a labeled anti-GPCR-B4 antibody. Alternatively, the labeling agentmay be a third moiety, such a secondary antibody, that specificallybinds to the antibody/GPCR-B4 complex (a secondary antibody is typicallyspecific to antibodies of the species from which the first antibody isderived). Other proteins capable of specifically binding immunoglobulinconstant regions, such as protein A or protein G may also be used as thelabel agent. These proteins exhibit a strong non-immunogenic reactivitywith immunoglobulin constant regions from a variety of species (see,e.g., Kronval et al., J. Immunol. 111:1401–1406 (1973); Akerstrom etal., J. Immunol. 135:2589–2542 (1985)). The labeling agent can bemodified with a detectable moiety, such as biotin, to which anothermolecule can specifically bind, such as streptavidin. A variety ofdetectable moieties are well known to those skilled in the art.

Throughout the assays, incubation and/or washing steps may be requiredafter each combination of reagents. Incubation steps can vary from about5 seconds to several hours, optionally from about 5 minutes to about 24hours. However, the incubation time will depend upon the assay format,antigen, volume of solution, concentrations, and the like. Usually, theassays will be carried out at ambient temperature, although they can beconducted over a range of temperatures, such as 10° C. to 40° C.

Non-competitive Assay Formats

Immunoassays for detecting GPCR-B4 in samples may be either competitiveor noncompetitive. Noncompetitive immunoassays are assays in which theamount of antigen is directly measured. In one preferred “sandwich”assay, for example, the anti-GPCR-B4 antibodies can be bound directly toa solid substrate on which they are immobilized. These immobilizedantibodies then capture GPCR-B4 present in the test sample. GPCR-B4 isthus immobilized is then bound by a labeling agent, such as a secondGPCR-B4 antibody bearing a label. Alternatively, the second antibody maylack a label, but it may, in turn, be bound by a labeled third antibodyspecific to antibodies of the species from which the second antibody isderived. The second or third antibody is typically modified with adetectable moiety, such as biotin, to which another moleculespecifically binds, e.g., streptavidin, to provide a detectable moiety.

Competitive Assay Formats

In competitive assays, the amount of GPCR-B4 present in the sample ismeasured indirectly by measuring the amount of a known, added(exogenous) GPCR-B4 displaced (competed away) from an anti-GPCR-B4antibody by the unknown GPCR-B4 present in a sample. In one competitiveassay, a known amount of GPCR-B4 is added to a sample and the sample isthen contacted with an antibody that specifically binds to GPCR-B4. Theamount of exogenous GPCR-B4 bound to the antibody is inverselyproportional to the concentration of GPCR-B4 present in the sample. In aparticularly preferred embodiment, the antibody is immobilized on asolid substrate. The amount of GPCR-B4 bound to the antibody may bedetermined either by measuring the amount of GPCR-B4 present in aGPCR-B4/antibody complex, or alternatively by measuring the amount ofremaining uncomplexed protein. The amount of GPCR-B4 may be detected byproviding a labeled GPCR-B4 molecule.

A hapten inhibition assay is another preferred competitive assay. Inthis assay the known GPCR-B4, is immobilized on a solid substrate. Aknown amount of anti-GPCR-B4 antibody is added to the sample, and thesample is then contacted with the immobilized GPCR-B4. The amount ofanti-GPCR-B4 antibody bound to the known immobilized GPCR-B4 isinversely proportional to the amount of GPCR-B4 present in the sample.Again, the amount of immobilized antibody may be detected by detectingeither the immobilized fraction of antibody or the fraction of theantibody that remains in solution. Detection may be direct where theantibody is labeled or indirect by the subsequent addition of a labeledmoiety that specifically binds to the antibody as described above.

Cross-reactivity Determinations

Immunoassays in the competitive binding format can also be used forcrossreactivity determinations. For example, a protein at leastpartially encoded by SEQ ID NOS:1–2, and 7 can be immobilized to a solidsupport. Proteins (e.g., GPCR-B4 proteins and homologs) are added to theassay that compete for binding of the antisera to the immobilizedantigen. The ability of the added proteins to compete for binding of theantisera to the immobilized protein is compared to the ability ofGPCR-B4 encoded by SEQ ID NO:1–2, or 7 to compete with itself. Thepercent crossreactivity for the above proteins is calculated, usingstandard calculations. Those antisera with less than 10% crossreactivitywith each of the added proteins listed above are selected and pooled.The cross-reacting antibodies are optionally removed from the pooledantisera by immunoabsorption with the added considered proteins, e.g.,distantly related homologs.

The immunoabsorbed and pooled antisera are then used in a competitivebinding immunoassay as described above to compare a second protein,thought to be perhaps an allele or polymorphic variant of GPCR-B4, tothe immunogen protein (i.e., GPCR-B4 of SEQ ID NOS:1–2 or 7). In orderto make this comparison, the two proteins are each assayed at a widerange of concentrations and the amount of each protein required toinhibit 50% of the binding of the antisera to the immobilized protein isdetermined. If the amount of the second protein required to inhibit 50%of binding is less than 10 times the amount of the protein encoded bySEQ ID NOS:1–2, or 7 that is required to inhibit 50% of binding, thenthe second protein is said to specifically bind to the polyclonalantibodies generated to a GPCR-B4 immunogen.

Other Assay Formats

Western blot (immunoblot) analysis is used to detect and quantify thepresence of GPCR-B4 in the sample. The technique generally comprisesseparating sample proteins by gel electrophoresis on the basis ofmolecular weight, transferring the separated proteins to a suitablesolid support, (such as a nitrocellulose filter, a nylon filter, orderivatized nylon filter), and incubating the sample with the antibodiesthat specifically bind GPCR-B4. The anti-GPCR-B4 antibodies specificallybind to the GPCR-B4 on the solid support. These antibodies may bedirectly labeled or alternatively may be subsequently detected usinglabeled antibodies (e.g., labeled sheep anti-mouse antibodies) thatspecifically bind to the anti-GPCR-B4 antibodies.

Other assay formats include liposome immunoassays (LIA), which useliposomes designed to bind specific molecules (e.g., antibodies) andrelease encapsulated reagents or markers. The released chemicals arethen detected according to standard techniques (see Monroe et al., Amer.Clin. Prod. Rev. 5:34–41 (1986)).

Reduction of Non-specific Binding

One of skill in the art will appreciate that it is often desirable tominimize non-specific binding in immunoassays. Particularly, where theassay involves an antigen or antibody immobilized on a solid substrateit is desirable to minimize the amount of non-specific binding to thesubstrate. Means of reducing such non-specific binding are well known tothose of skill in the art. Typically, this technique involves coatingthe substrate with a proteinaceous composition. In particular, proteincompositions such as bovine serum albumin (BSA), nonfat powdered milk,and gelatin are widely used with powdered milk being most preferred.

Labels

The particular label or detectable group used in the assay is not acritical aspect of the invention, as long as it does not significantlyinterfere with the specific binding of the antibody used in the assay.The detectable group can be any material having a detectable physical orchemical property. Such detectable labels have been well-developed inthe field of immunoassays and, in general, most any label useful in suchmethods can be applied to the present invention. Thus, a label is anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Useful labels inthe present invention include magnetic beads (e.g., DYNABEADS™),fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red,rhodamine, and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or³²P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase andothers commonly used in an ELISA), and colorimetric labels such ascolloidal gold or colored glass or plastic beads (e.g., polystyrene,polypropylene, latex, etc.).

The label may be coupled directly or indirectly to the desired componentof the assay according to methods well known in the art. As indicatedabove, a wide variety of labels may be used, with the choice of labeldepending on sensitivity required, ease of conjugation with thecompound, stability requirements, available instrumentation, anddisposal provisions.

Non-radioactive labels are often attached by indirect means. Generally,a ligand molecule (e.g., biotin) is covalently bound to the molecule.The ligand then binds to another molecules (e.g., streptavidin)molecule, which is either inherently detectable or covalently bound to asignal system, such as a detectable enzyme, a fluorescent compound, or achemiluminescent compound. The ligands and their targets can be used inany suitable combination with antibodies that recognize GPCR-B4, orsecondary antibodies that recognize anti-GPCR-B4.

The molecules can also be conjugated directly to signal generatingcompounds, e.g., by conjugation with an enzyme or fluorophore. Enzymesof interest as labels will primarily be hydrolases, particularlyphosphatases, esterases and glycosidases, or oxidotases, particularlyperoxidases. Fluorescent compounds include fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.Chemiluminescent compounds include luciferin, and2,3-dihydrophthalazinediones, e.g., luminol. For a review of variouslabeling or signal producing systems that may be used, see U.S. Pat. No.4,391,904.

Means of detecting labels are well known to those of skill in the art.Thus, for example, where the label is a radioactive label, means fordetection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence may bedetected visually, by means of photographic film, by the use ofelectronic detectors such as charge coupled devices (CCDs) orphotomultipliers and the like. Similarly, enzymatic labels may bedetected by providing the appropriate substrates for the enzyme anddetecting the resulting reaction product. Finally simple colorimetriclabels may be detected simply by observing the color associated with thelabel. Thus, in various dipstick assays, conjugated gold often appearspink, while various conjugated beads appear the color of the bead.

Some assay formats do not require the use of labeled components. Forinstance, agglutination assays can be used to detect the presence of thetarget antibodies. In this case, antigen-coated particles areagglutinated by samples comprising the target antibodies. In thisformat, none of the components need be labeled and the presence of thetarget antibody is detected by simple visual inspection.

VI. Assays for Modulators of GPCR-B4

A. Assays for GPCR-B4 Activity

GPCR-B4 and its alleles and polymorphic variants are G-protein coupledreceptors that participate in taste transduction. The activity ofGPCR-B4 polypeptides can be assessed using a variety of in vitro and invivo assays to determine functional, chemical, and physical effects,e.g., measuring ligand binding (e.g., radioactive ligand binding),second messengers (e.g., cAMP, cGMP, IP₃, DAG, or Ca²⁺), ion flux,phosphorylation levels, transcription levels, neurotransmitter levels,and the like. Furthermore, such assays can be used to test forinhibitors and activators of GPCR-B4. Modulators can also be geneticallyaltered versions of GPCR-B4. Such modulators of taste transductionactivity are useful for customizing taste.

The GPCR-B4 of the assay will be selected from a polypeptide having asequence of SEQ ID NOS:1–2, or 7 or conservatively modified variantthereof. Alternatively, the GPCR-B4 of the assay will be derived from aeukaryote and include an amino acid subsequence having amino acidsequence identity SEQ ID NOS:1–2, or 7. Generally, the amino acidsequence identity will be at least 70%, optionally at least 85%,optionally at least 90–95%. Optionally, the polypeptide of the assayswill comprise a domain of GPCR-B4, such as an extracellular domain,transmembrane domain, cytoplasmic domain, ligand binding domain, subunitassociation domain, active site, and the like. Either GPCR-B4 or adomain thereof can be covalently linked to a heterologous protein tocreate a chimeric protein used in the assays described herein.

Modulators of GPCR-B4 activity are tested using GPCR-B4 polypeptides asdescribed above, either recombinant or naturally occurring. The proteincan be isolated, expressed in a cell, expressed in a membrane derivedfrom a cell, expressed in tissue or in an animal, either recombinant ornaturally occurring. For example, tongue slices, dissociated cells froma tongue, transformed cells, or membranes can b used. Modulation istested using one of the in vitro or in vivo assays described herein.Taste transduction can also be examined in vitro with soluble or solidstate reactions, using a chimeric molecule such as an extracellulardomain of a receptor covalently linked to a heterologous signaltransduction domain, or a heterologous extracellular domain covalentlylinked to the transmembrane and or cytoplasmic domain of a receptor.Furthermore, ligand-binding domains of the protein of interest can beused in vitro in soluble or solid state reactions to assay for ligandbinding.

Ligand binding to GPCR-B4, a domain, or chimeric protein can be testedin solution, in a bilayer membrane, attached to a solid phase, in alipid monolayer, or in vesicles. Binding of a modulator can be testedusing, e.g., changes in spectroscopic characteristics (e.g.,fluorescence, absorbance, refractive index) hydrodynamic (e.g., shape),chromatographic, or solubility properties.

Receptor-G-protein interactions can also be examined. For example,binding of the G-protein to the receptor or its release from thereceptor can be examined. For example, in the absence of GTP, anactivator will lead to the formation of a tight complex of a G protein(all three subunits) with the receptor. This complex can be detected ina variety of ways, as noted above. Such an assay can be modified tosearch for inhibitors. Add an activator to the receptor and G protein inthe absence of GTP, form a tight complex, and then screen for inhibitorsby looking at dissociation of the receptor-G protein complex. In thepresence of GTP, release of the alpha subunit of the G protein from theother two G protein subunits serves as a criterion of activation.

An activated or inhibited G-protein will in turn alter the properties oftarget enzymes, channels, and other effector proteins. The classicexamples are the activation of cGMP phosphodiesterase by transducin inthe visual system, adenylate cyclase by the stimulatory G-protein,phospholipase C by Gq and other cognate G proteins, and modulation ofdiverse channels by Gi and other G proteins. Downstream consequences canalso be examined such as generation of diacyl glycerol and IP3 byphospholipase C, and in turn, for calcium mobilization by IP3.

Activated GPCR receptors become substrates for kinases thatphosphorylate the C-terminal tail of the receptor (and possibly othersites as well). Thus, activators will promote the transfer of ³²P fromgamma-labeled GTP to the receptor, which can be assayed with ascintillation counter. The phosphorylation of the C-terminal tail willpromote the binding of arrestin-like proteins and will interfere withthe binding of G-proteins. The kinase/arrestin pathway plays a key rolein the desensitization of many GPCR receptors. For example, compoundsthat modulate the duration a taste receptor stays active would be usefulas a means of prolonging a desired taste or cutting off an unpleasantone. For a general review of GPCR signal transduction and methods ofassaying signal transduction, see, e.g., Methods in Enzymology, vols.237 and 238 (1994) and volume 96 (1983); Bourne et al., Nature10:349:117–27 (1991); Bourne et al., Nature 348:125–32 (1990); Pitcheret al., Annu. Rev. Biochem. 67:653–92 (1998).

Samples or assays that are treated with a potential GPCR-B4 inhibitor oractivator are compared to control samples without the test compound, toexamine the extent of modulation. Control samples (untreated withactivators or inhibitors) are assigned a relative GPCR-B4 activity valueof 100. Inhibition of GPCR-B4 is achieved when the GPCR-B4 activityvalue relative to the control is about 90%, optionally 50%, optionally25–0%. Activation of GPCR-B4 is achieved when the GPCR-B4 activity valuerelative to the control is 110%, optionally 150%, 200–500%, or1000–2000%.

Changes in ion flux may be assessed by determining changes inpolarization (i.e., electrical potential) of the cell or membraneexpressing GPCR-B4. One means to determine changes in cellularpolarization is by measuring changes in current (thereby measuringchanges in polarization) with voltage-clamp and patch-clamp techniques,e.g., the “cell-attached” mode, the “inside-out” mode, and the “wholecell” mode (see, e.g., Ackerman et al., New Engl. J. Med. 336:1575–1595(1997)). Whole cell currents are conveniently determined using thestandard methodology (see, e.g., Hamil et al., PFlugers. Archiv. 391:85(1981). Other known assays include: radiolabeled ion flux assays andfluorescence assays using voltage-sensitive dyes (see, e.g.,Vestergarrd-Bogind et al., J. Membrane Biol. 88:67–75 (1988); Gonzales &Tsien, Chem. Biol. 4:269–277 (1997); Daniel et al., J. Pharmacol. Meth.25:185–193 (1991); Holevinsky et al., J. Membrane Biology 137:59–70(1994)). Generally, the compounds to be tested are present in the rangefrom 1 pM to 100 mM.

The effects of the test compounds upon the function of the polypeptidescan be measured by examining any of the parameters described above. Anysuitable physiological change that affects GPCR activity can be used toassess the influence of a test compound on the polypeptides of thisinvention. When the functional consequences are determined using intactcells or animals, one can also measure a variety of effects such astransmitter release, hormone release, transcriptional changes to bothknown and uncharacterized genetic markers (e.g., northern blots),changes in cell metabolism such as cell growth or pH changes, andchanges in intracellular second messengers such as Ca²⁺, IP3 or cAMP.

Preferred assays for G-protein coupled receptors include cells that areloaded with ion or voltage sensitive dyes to report receptor activity.Assays for determining activity of such receptors can also use knownagonists and antagonists for other G-protein coupled receptors asnegative or positive controls to assess activity of tested compounds. Inassays for identifying modulatory compounds (e.g., agonists,antagonists), changes in the level of ions in the cytoplasm or membranevoltage will be monitored using an ion sensitive or membrane voltagefluorescent indicator, respectively. Among the ion-sensitive indicatorsand voltage probes that may be employed are those disclosed in theMolecular Probes 1997 Catalog. For G-protein coupled receptors,promiscuous G-proteins such as Gα15 and Gα16 can be used in the assay ofchoice (Wilkie et al., Proc. Nat'l Acad. Sci. USA 88:10049–10053(1991)). Such promiscuous G-proteins allow coupling of a wide range ofreceptors.

Receptor activation typically initiates subsequent intracellular events,e.g., increases in second messengers such as IP3, which releasesintracellular stores of calcium ions. Activation of some G-proteincoupled receptors stimulates the formation of inositol triphosphate(IP3) through phospholipase C-mediated hydrolysis ofphosphatidylinositol (Berridge & Irvine, Nature 312:315–21 (1984)). IP3in turn stimulates the release of intracellular calcium ion stores.Thus, a change in cytoplasmic calcium ion levels, or a change in secondmessenger levels such as IP3 can be used to assess G-protein coupledreceptor function. Cells expressing such G-protein coupled receptors mayexhibit increased cytoplasmic calcium levels as a result of contributionfrom both intracellular stores and via activation of ion channels, inwhich case it may be desirable although not necessary to conduct suchassays in calcium-free buffer, optionally supplemented with a chelatingagent such as EGTA, to distinguish fluorescence response resulting fromcalcium release from internal stores.

Other assays can involve determining the activity of receptors which,when activated, result in a change in the level of intracellular cyclicnucleotides, e.g., cAMP or cGMP, by activating or inhibiting enzymessuch as adenylate cyclase. There are cyclic nucleotide-gated ionchannels, e.g., rod photoreceptor cell channels and olfactory neuronchannels that are permeable to cations upon activation by binding ofcAMP or cGMP (see, e.g., Altenhofen et al., Proc. Natl. Acad. Sci.U.S.A. 88:9868–9872 (1991) and Dhallan et al., Nature 347:184–187(1990)). In cases where activation of the receptor results in a decreasein cyclic nucleotide levels, it may be preferable to expose the cells toagents that increase intracellular cyclic nucleotide levels, e.g.,forskolin, prior to adding a receptor-activating compound to the cellsin the assay. Cells for this type of assay can be made byco-transfection of a host cell with DNA encoding a cyclicnucleotide-crated ion channel, GPCR phosphatase and DNA encoding areceptor (e.g., certain glutamate receptors, muscarinic acetylcholinereceptors, dopamine receptors, serotonin receptors, and the like),which, when activated, causes a change in cyclic nucleotide levels inthe cytoplasm.

In a preferred embodiment, GPCR-B4 activity is measured by expressingGPCR-B4 in a heterologous cell with a promiscuous G-protein that linksthe receptor to a phospholipase C signal transduction pathway (seeOffermanns & Simon, J. Biol. Chem. 270:15175–15180 (1995); see alsoExample II). Optionally the cell line is HEK-293 (which does notnaturally express GPCR-B4) and the promiscuous G-protein is Gα15(Offermanns & Simon, supra). Modulation of taste transduction is assayedby measuring changes in intracellular Ca²⁺ levels, which change inresponse to modulation of the GPCR-B4 signal transduction pathway viaadministration of a molecule that associates with GPCR-B4. Changes inCa²⁺ levels are optionally measured using fluorescent Ca²⁺ indicatordyes and fluorometric imaging.

In one embodiment, the changes in intracellular cAMP or cGMP can bemeasured using immunoassays. The method described in Offermanns & Simon,J. Biol. Chem. 270:15175–15180 (1995) may be used to determine the levelof cAMP. Also, the method described in Felley-Bosco et al., Am. J. Resp.Cell and Mol. Biol. 11:159–164 (1994) may be used to determine the levelof cGMP. Further, an assay kit for measuring cAMP and/or cGMP isdescribed in U.S. Pat. No. 4,115,538, herein incorporated by reference.

In another embodiment, phosphatidyl inositol (PI) hydrolysis can beanalyzed according to U.S. Pat. No. 5,436,128, herein incorporated byreference. Briefly, the assay involves labeling of cells with³H-myoinositol for 48 or more hrs. The labeled cells are treated with atest compound for one hour. The treated cells are lysed and extracted inchloroform-methanol-water after which the inositol phosphates wereseparated by ion exchange chromatography and quantified by scintillationcounting. Fold stimulation is determined by calculating the ratio of cpmin the presence of agonist to cpm in the presence of buffer control.Likewise, fold inhibition is determined by calculating the ratio of cpmin the presence of antagonist to cpm in the presence of buffer control(which may or may not contain an agonist).

In another embodiment, transcription levels can be measured to assessthe effects of a test compound on signal transduction. A host cellcontaining the protein of interest is contacted with a test compound fora sufficient time to effect any interactions, and then the level of geneexpression is measured. The amount of time to effect such interactionsmay be empirically determined, such as by running a time course andmeasuring the level of transcription as a function of time. The amountof transcription may be measured by using any method known to those ofskill in the art to be suitable. For example, mRNA expression of theprotein of interest may be detected using northern blots or theirpolypeptide products may be identified using immunoassays.Alternatively, transcription based assays using reporter gene may beused as described in U.S. Pat. No. 5,436,128, herein incorporated byreference. The reporter genes can be, e.g., chloramphenicolacetyltransferase, firefly luciferase, bacterial luciferase,β-galactosidase and alkaline phosphatase. Furthermore, the protein ofinterest can be used as an indirect reporter via attachment to a secondreporter such as green fluorescent protein (see, e.g., Mistili &Spector, Nature Biotechnology 15:961–964 (1997)).

The amount of transcription is then compared to the amount oftranscription in either the same cell in the absence of the testcompound, or it may be compared with the amount of transcription in asubstantially identical cell that lacks the protein of interest. Asubstantially identical cell may be derived from the same cells fromwhich the recombinant cell was prepared but which had not been modifiedby introduction of heterologous DNA. Any difference in the amount oftranscription indicates that the test compound has in some manneraltered the activity of the protein of interest.

B. Modulators

The compounds tested as modulators of GPCR-B4 can be any small chemicalcompound, or a biological entity, such as a protein, sugar, nucleic acidor lipid. Alternatively, modulators can be genetically altered versionsof GPCR-B4. Typically, test compounds will be small chemical moleculesand peptides. Essentially any chemical compound can be used as apotential modulator or ligand in the assays of the invention, althoughmost often compounds can be dissolved in aqueous or organic (especiallyDMSO-based) solutions are used. The assays are designed to screen largechemical libraries by automating the assay steps and providing compoundsfrom any convenient source to assays, which are typically run inparallel (e.g., in microtiter formats on microtiter plates in roboticassays). It will be appreciated that there are many suppliers ofchemical compounds, including Sigma (St. Louis, Mo.), Aldrich (St.Louis, Mo.), Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-BiochemicaAnalytika (Buchs Switzerland) and the like.

In one preferred embodiment, high throughput screening methods involveproviding a combinatorial chemical or peptide library containing a largenumber of potential therapeutic compounds (potential modulator or ligandcompounds). Such “combinatorial chemical libraries” or “ligandlibraries” are then screened in one or more assays, as described herein,to identify those library members (particular chemical species orsubclasses) that display a desired characteristic activity. Thecompounds thus identified can serve as conventional “lead compounds” orcan themselves be used as potential or actual therapeutics.

A combinatorial chemical library is a collection of diverse chemicalcompounds generated by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks” such asreagents. For example, a linear combinatorial chemical library such as apolypeptide library is formed by combining a set of chemical buildingblocks (amino acids) in every possible way for a given compound length(i.e., the number of amino acids in a polypeptide compound). Millions ofchemical compounds can be synthesized through such combinatorial mixingof chemical building blocks.

Preparation and screening of combinatorial chemical libraries is wellknown to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, e.g.,U.S. Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487–493(1991) and Houghton et al., Nature 354:84–88 (1991)). Other chemistriesfor generating chemical diversity libraries can also be used. Suchchemistries include, but are not limited to: peptoids (e.g., PCTPublication No. WO 91/19735), encoded peptides (e.g., PCT Publication WO93/20242), random bio-oligomers (e.g., PCT Publication No. WO 92/00091),benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomers such ashydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc. Nat.Acad. Sci. USA 90:6909–6913 (1993)), vinylogous polypeptides (Hagiharaet al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidalpeptidomimetics with glucose scaffolding (Hirschmann et al., J. Amer.Chem. Soc. 114:9217–9218 (1992)), analogous organic syntheses of smallcompound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)),oligocarbamates (Cho et al., Science 261:1303 (1993)), and/or peptidylphosphonates (Campbell et al., J. Org. Chem. 59:658 (1994)), nucleicacid libraries (see Ausubel, Berger and Sambrook, all supra), peptidenucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083), antibodylibraries (see, e.g., Vaughn et al., Nature Biotechnology, 14(3):309–314(1996) and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang etal., Science, 274:1520–1522 (1996) and U.S. Pat. No. 5,593,853), smallorganic molecule libraries (see, e.g., benzodiazepines, Baum C&EN, Jan18, page 33 (1993); isoprenoids, U.S. Pat. No. 5,569,588;thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974;pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholinocompounds, U.S. Pat. No. 5,506,337; benzodiazepines, U.S. Pat. No.5,288,514, and the like).

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, LouisvilleKy., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, FosterCity, Calif., 9050 Plus, Millipore, Bedford, Mass.). In addition,numerous combinatorial libraries are themselves commercially available(see, e.g., ComGenex, Princeton, N.J., Tripos, Inc., St. Louis, Mo., 3DPharmaceuticals, Exton, Pa., Martek Biosciences, Columbia, Md., etc.).

C. Solid State and Soluble High Throughput Assays

In one embodiment the invention provide soluble assays using moleculessuch as a domain such as ligand binding domain, an extracellular domain,a transmembrane domain (e.g., one comprising seven transmembrane regionsand cytosolic loops), the transmembrane domain and a cytoplasmic domain,an active site, a subunit association region, etc.; a domain that iscovalently linked to a heterologous protein to create a chimericmolecule; GPCR-B4; or a cell or tissue expressing GPCR-B4, eithernaturally occurring or recombinant. In another embodiment, the inventionprovides solid phase based in vitro assays in a high throughput format,where the domain, chimeric molecule, GPCR-B4, or cell or tissueexpressing GPCR-B4 is attached to a solid phase substrate.

In the high throughput assays of the invention, it is possible to screenup to several thousand different modulators or ligands in a single day.In particular, each well of a microtiter plate can be used to run aseparate assay against a selected potential modulator, or, ifconcentration or incubation time effects are to be observed, every 5–10wells can test a single modulator. Thus, a single standard microtiterplate can assay about 100 (e.g., 96) modulators. If 1536 well plates areused, then a single plate can easily assay from about 100- about 1500different compounds. It is possible to assay several different platesper day; assay screens for up to about 6,000–20,000 different compoundsis possible using the integrated systems of the invention. Morerecently, microfluidic approaches to reagent manipulation have beendeveloped, e.g., by Caliper Technologies (Palo Alto, Calif.).

The molecule of interest can be bound to the solid state component,directly or indirectly, via covalent or non covalent linkage e.g., via atag. The tag can be any of a variety of components. In general, amolecule which binds the tag (a tag binder) is fixed to a solid support,and the tagged molecule of interest (e.g., the taste transductionmolecule of interest) is attached to the solid support by interaction ofthe tag and the tag binder.

A number of tags and tag binders can be used, based upon known molecularinteractions well described in the literature. For example, where a taghas a natural binder, for example, biotin, protein A, or protein G, itcan be used in conjunction with appropriate tag binders (avidin,streptavidin, neutravidin, the Fc region of an immunoglobulin, etc.)Antibodies to molecules with natural binders such as biotin are alsowidely available and appropriate tag binders; see, SIGMA Immunochemicals1998 catalogue SIGMA, St. Louis Mo.).

Similarly, any haptenic or antigenic compound can be used in combinationwith an appropriate antibody to form a tag/tag binder pair. Thousands ofspecific antibodies are commercially available and many additionalantibodies are described in the literature. For example, in one commonconfiguration, the tag is a first antibody and the tag binder is asecond antibody which recognizes the first antibody. In addition toantibody-antigen interactions, receptor-ligand interactions are alsoappropriate as tag and tag-binder pairs. For example, agonists andantagonists of cell membrane receptors (e.g., cell receptor-ligandinteractions such as transferrin, c-kit, viral receptor ligands,cytokine receptors, chemokine receptors, interleukin receptors,immunoglobulin receptors and antibodies, the cadherein family, theintegrin family, the selectin family, and the like; see, e.g., Pigott &Power, The Adhesion Molecule Facts Book I (1993). Similarly, toxins andvenoms, viral epitopes, hormones (e.g., opiates, steroids, etc.),intracellular receptors (e.g. which mediate the effects of various smallligands, including steroids, thyroid hormone, retinoids and vitamin D;peptides), drugs, lectins, sugars, nucleic acids (both linear and cyclicpolymer configurations), oligosaccharides, proteins, phospholipids andantibodies can all interact with various cell receptors.

Synthetic polymers, such as polyurethanes, polyesters, polycarbonates,polyureas, polyamides, polyethyleneimines, polyarylene sulfides,polysiloxanes, polyimides, and polyacetates can also form an appropriatetag or tag binder. Many other tag/tag binder pairs are also useful inassay systems described herein, as would be apparent to one of skillupon review of this disclosure.

Common linkers such as peptides, polyethers, and the like can also serveas tags, and include polypeptide sequences, such as poly gly sequencesof between about 5 and 200 amino acids. Such flexible linkers are knownto persons of skill in the art. For example, poly(ethelyne glycol)linkers are available from Shearwater Polymers, Inc. Huntsville, Ala.These linkers optionally have amide linkages, sulfhydryl linkages, orheterofunctional linkages.

Tag binders are fixed to solid substrates using any of a variety ofmethods currently available. Solid substrates are commonly derivatizedor functionalized by exposing all or a portion of the substrate to achemical reagent which fixes a chemical group to the surface which isreactive with a portion of the tag binder. For example, groups which aresuitable for attachment to a longer chain portion would include amines,hydroxyl, thiol, and carboxyl groups. Aminoalkylsilanes andhydroxyalkylsilanes can be used to functionalize a variety of surfaces,such as glass surfaces. The construction of such solid phase biopolymerarrays is well described in the literature. See, e.g., Merrifield, J.Am. Chem. Soc. 85:2149–2154 (1963) (describing solid phase synthesis of,e.g., peptides); Geysen et al., J. Immun. Meth. 102:259–274 (1987)(describing synthesis of solid phase components on pins); Frank &Doring, Tetrahedron 44:60316040 (1988) (describing synthesis of variouspeptide sequences on cellulose disks); Fodor et al., Science,251:767–777 (1991); Sheldon et al., Clinical Chemistry 39(4):718–719(1993); and Kozal et al., Nature Medicine 2(7):753759 (1996) (alldescribing arrays of biopolymers fixed to solid substrates).Non-chemical approaches for fixing tag binders to substrates includeother common methods, such as heat, cross-linking by UV radiation, andthe like.

D. Computer-based Assays

Yet another assay for compounds that modulate GPCR-B4 activity involvescomputer assisted drug design, in which a computer system is used togenerate a three-dimensional structure of GPCR-B4 based on thestructural information encoded by the amino acid sequence. The inputamino acid sequence interacts directly and actively with apreestablished algorithm in a computer program to yield secondary,tertiary, and quaternary structural models of the protein. The models ofthe protein structure are then examined to identify regions of thestructure that have the ability to bind, e.g., ligands. These regionsare then used to identify ligands that bind to the protein.

The three-dimensional structural model of the protein is generated byentering protein amino acid sequences of at least 10 amino acid residuesor corresponding nucleic acid sequences encoding a GPCR-B4 polypeptideinto the computer system. The amino acid sequence of the polypeptide ofthe nucleic acid encoding the polypeptide is selected from the groupconsisting of SEQ ID NOS:1–2, or 7 or SEQ ID NOS:3–4, or 8 andconservatively modified versions thereof. The amino acid sequencerepresents the primary sequence or subsequence of the protein, whichencodes the structural information of the protein. At least 10 residuesof the amino acid sequence (or a nucleotide sequence encoding 10 aminoacids) are entered into the computer system from computer keyboards,computer readable substrates that include, but are not limited to,electronic storage media (e.g., magnetic diskettes, tapes, cartridges,and chips), optical media (e.g., CD ROM), information distributed byinternet sites, and by RAM. The three-dimensional structural model ofthe protein is then generated by the interaction of the amino acidsequence and the computer system, using software known to those of skillin the art.

The amino acid sequence represents a primary structure that encodes theinformation necessary to form the secondary, tertiary and quaternarystructure of the protein of interest. The software looks at certainparameters encoded by the primary sequence to generate the structuralmodel. These parameters are referred to as “energy terms,” and primarilyinclude electrostatic potentials, hydrophobic potentials, solventaccessible surfaces, and hydrogen bonding. Secondary energy termsinclude van der Waals potentials. Biological molecules form thestructures that minimize the energy terms in a cumulative fashion. Thecomputer program is therefore using these terms encoded by the primarystructure or amino acid sequence to create the secondary structuralmodel.

The tertiary structure of the protein encoded by the secondary structureis then formed on the basis of the energy terms of the secondarystructure. The user at this point can enter additional variables such aswhether the protein is membrane bound or soluble, its location in thebody, and its cellular location, e.g., cytoplasmic, surface, or nuclear.These variables along with the energy terms of the secondary structureare used to form the model of the tertiary structure. In modeling thetertiary structure, the computer program matches hydrophobic faces ofsecondary structure with like, and hydrophilic faces of secondarystructure with like.

Once the structure has been generated, potential ligand binding regionsare identified by the computer system. Three-dimensional structures forpotential ligands are generated by entering amino acid or nucleotidesequences or chemical formulas of compounds, as described above. Thethree-dimensional structure of the potential ligand is then compared tothat of the GPCR-B4 protein to identify ligands that bind to GPCR-B4.Binding affinity between the protein and ligands is determined usingenergy terms to determine which ligands have an enhanced probability ofbinding to the protein.

Computer systems are also used to screen for mutations, polymorphicvariants, alleles and interspecies homologs of GPCR-B4 genes. Suchmutations can be associated with disease states or genetic traits. Asdescribed above, GeneChip™ and related technology can also be used toscreen for mutations, polymorphic variants, alleles and interspecieshomologs. Once the variants are identified, diagnostic assays can beused to identify patients having such mutated genes. Identification ofthe mutated GPCR-B4 genes involves receiving input of a first nucleicacid or amino acid sequence encoding GPCR-B4, selected from the groupconsisting of SEQ ID NOS:1–2, and 7, or SEQ ID NOS:3–4, and 8 andconservatively modified versions thereof. The sequence is entered intothe computer system as described above. The first nucleic acid or aminoacid sequence is then compared to a second nucleic acid or amino acidsequence that has substantial identity to the first sequence. The secondsequence is entered into the computer system in the manner describedabove. Once the first and second sequences are compared, nucleotide oramino acid differences between the sequences are identified. Suchsequences can represent allelic differences in GPCR-B4 genes, andmutations associated with disease states and genetic traits.

VIII. Kits

GPCR-B4 and its homologs are a useful tool for identifying tastereceptor cells, for forensics and paternity determinations, and forexamining taste transduction. GPCR-B4 specific reagents thatspecifically hybridize to GPCR-B4 nucleic acid, such as GPCR-B4 probesand primers, and GPCR-B4 specific reagents that specifically bind to theGPCR-B4 protein, e.g., GPCR-B4 antibodies are used to examine taste cellexpression and taste transduction regulation.

Nucleic acid assays for the presence of GPCR-B4 DNA and RNA in a sampleinclude numerous techniques are known to those skilled in the art, suchas Southern analysis, northern analysis, dot blots, RNase protection, S1analysis, amplification techniques such as PCR and LCR, and in situhybridization. In in situ hybridization, for example, the target nucleicacid is liberated from its cellular surroundings in such as to beavailable for hybridization within the cell while preserving thecellular morphology for subsequent interpretation and analysis (seeExample I). The following articles provide an overview of the art of insitu hybridization: Singer et al., Biotechniques 4:230–250 (1986); Haaseet al., Methods in Virology, vol. VII, pp.189–226 (1984); and NucleicAcid Hybridization: A Practical Approach (Hames et al., eds. 1987). Inaddition, GPCR-B4 protein can be detected with the various immunoassaytechniques described above. The test sample is typically compared toboth a positive control (e.g., a sample expressing recombinant GPCR-B4)and a negative control.

The present invention also provides for kits for screening formodulators of GPCR-B4. Such kits can be prepared from readily availablematerials and reagents. For example, such kits can comprise any one ormore of the following materials: GPCR-B4, reaction tubes, andinstructions for testing GPCR-B4 activity. Optionally, the kit containsbiologically active GPCR-B4. A wide variety of kits and components canbe prepared according to the present invention, depending upon theintended user of the kit and the particular needs of the user.

IX. Administration and Pharmaceutical Compositions

Taste modulators can be administered directly to the mammalian subjectfor modulation of taste in vivo. Administration is by any of the routesnormally used for introducing a modulator compound into ultimate contactwith the tissue to be treated, optionally the tongue or mouth. The tastemodulators are administered in any suitable manner, optionally withpharmaceutically acceptable carriers. Suitable methods of administeringsuch modulators are available and well known to those of skill in theart, and, although more than one route can be used to administer aparticular composition, a particular route can often provide a moreimmediate and more effective reaction than another route.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositions of thepresent invention (see, e.g., Remington's Pharmaceutical Sciences,17^(th) ed. 1985)).

The taste modulators, alone or in combination with other suitablecomponents, can be made into aerosol formulations (i.e., they can be“nebulized”) to be administered via inhalation. Aerosol formulations canbe placed into pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like.

Formulations suitable for administration include aqueous and non-aqueoussolutions, isotonic sterile solutions, which can contain antioxidants,buffers, bacteriostats, and solutes that render the formulationisotonic, and aqueous and non-aqueous sterile suspensions that caninclude suspending agents, solubilizers, thickening agents, stabilizers,and preservatives. In the practice of this invention, compositions canbe administered, for example, by orally, topically, intravenously,intraperitoneally, intravesically or intrathecally. Optionally, thecompositions are administered orally or nasally. The formulations ofcompounds can be presented in unit-dose or multi-dose sealed containers,such as ampules and vials. Solutions and suspensions can be preparedfrom sterile powders, granules, and tablets of the kind previouslydescribed. The modulators can also be administered as part a of preparedfood or drug.

The dose administered to a patient, in the context of the presentinvention should be sufficient to effect a beneficial response in thesubject over time. The dose will be determined by the efficacy of theparticular taste modulators employed and the condition of the subject,as well as the body weight or surface area of the area to be treated.The size of the dose also will be determined by the existence, nature,and extent of any adverse side-effects that accompany the administrationof a particular compound or vector in a particular subject.

In determining the effective amount of the modulator to be administeredin a physician may evaluate circulating plasma levels of the modulator,modulator toxicities,, and the production of anti-modulator antibodies.In general, the dose equivalent of a modulator is from about 1 ng/kg to10 mg/kg for a typical subject.

For administration, taste modulators of the present invention can beadministered at a rate determined by the LD-50 of the modulator, and theside-effects of the inhibitor at various concentrations, as applied tothe mass and overall health of the subject. Administration can beaccomplished via single or divided doses.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

EXAMPLES

The following examples are provided by way of illustration only and notby way of limitation. Those of skill in the art will readily recognize avariety of noncritical parameters that could be changed or modified toyield essentially similar results.

Example I

Cloning and Expression of GPCR-B4

cDNA libraries made from rat circumvallate and fungiform single cellswas used isolate the GPCR nucleic acids of the invention.

Single foliate and fungiform papillae were isolated from the rat tongue(10 papillae each) and first strand cDNA was prepared from each papillaeusing single-cell library construction methods (see, e.g., Bernhardt etal., J. Physiol. 490:325–336 (1996); Dulac & Axel, Cell 83:195–206(1995)). 20 different cDNA populations were assayed for those positivefor a taste receptor marker (TCP #1, also known as clone 27–59; seepatent application Ser. No. 60/094,464, filed Jul. 28, 1998) to ensurethat the cDNA was from taste receptor cells. The cDNAs were alsoscreened with GPCR-B3, a G-protein coupled receptor clone (see U.S. Ser.No. 60/094,465, filed Jul. 28, 1998). Three positive papillae wereidentified and used as a source of cDNA for PCR amplifications usingdegenerate primers designed to encode motifs highly conserved amongstVR/mGluR/CaST/GPCR-B3 receptors. Preferred primers came from the areabetween transmembrane domains 6 and 7: [Y/N]FNEAK (SEQ ID NO:9) andPKCY[I/V]I (SEQ ID NO:10). Degenerate PCR products were subcloned into aBluescript vector as HindIII fragments, and 52 PCR products weresequences. Twenty of these products corresponded to GPCR-B3. 8 of theproducts encoded a novel GPCR-B4 sequence.

Mouse interspecies homologs of GPCR-B4 were isolated using the ratGPCR-B4 clones as probes for genomic and cDNA libraries. The nucleotideand amino acid sequences of GPCR-B4 are provided, respectively, in SEQID NO:1–2, and 7 and SEQ ID NO:3–4, and 8.

Taste cell specific expression of GPCR-B4 is confirmed using the clonesas probes for in situ hybridization to tongue tissue sections. Allclones demonstrate specific or preferential expression in taste buds.

Example II

GPCR is a Taste Transduction Receptor

The distinctive topographic distribution of GPCR-B4 and the behavioralrepresentation of bitter transduction suggest a correlation between thesites of expression of B4 (circumvallate papillae, but not fungiform orgeschmackstreifen) and bitter sensitivity. To determine the ligandselectivity of GPCR-B4, expression in heterologous cells was used. Oneissue for GPCR expression in heterologous cells is determining how tocouple the GPCR to a G-protein and an appropriate signaling pathway. Inthis example, the G-protein subunit Gα15 was used, which promiscuouslycouples a wide range of GPCRs to the phospholipase C-mediated signalingpathway (Offermans & Simon, J. Biol. Chem. 270:15175–15180 (1995)).Consequently, receptor activity can be effectively measured by recordingligand-induced changes in [Ca²⁺]_(i) using fluorescent Ca²⁺-indicatordyes and fluorometric imaging.

To insure expression of GPCR-B4 in the plasma membrane, a variety ofcell lines and expression vectors were tested. As a control for thesestudies, the cells were transfected with a mammalian γ-opiod receptor;this receptor does not normally couple to PLC, so all agonist-inducedchanges in [Ca²⁺]_(i) reflect coupling through Gα15. An HEK-293 lineexpressing SV40 T-antigen was co-transfected with a TK-Gα15, CMV-γ-opiodand a pEAK-10 episomal vector (Edge Biosystems) containing aEF1a-[B4-GPCR] construct. Transfection efficiencies were determinedusing CMV-GFP constructs. Control cells expressing γ-opiod/Gα15 respondrobustly to DAMGO (a γ-opiod agonist), but do not respond to sweet orbitter tastants, or unrelated agonists (data not shown). These responsesare dependent on Gα15, and have the appropriate temporal resolution,with rapid onset following application of the stimulus. Notably, cellsexpressing B4/Gα15 or B4/Gα15/γ-opiod respond to the well characterizedbitter tastant phenylthiocarbamide (PTC), but not to any of a number ofnatural or artificial sweeteners. This activity is entirely B4 receptordependent, and occurs at physiologically relevant concentrations of PTC(300 μM-5 mM).

These results suggest GPCR-B4 is involved in bitter taste transduction,and provide an experimentally tractable system for future experiments,including studies of tastant specificity and selectivity, the definitionof the native bitter signaling pathway, and perhaps understanding themolecular basis of human psychophysical studies demonstrating dramaticdifferences in PTC tasting between “tasters” and “non-tasters.”

1. An isolated sensory transduction G-protein coupled receptor, thereceptor comprising greater than about 90% amino acid sequence identityto the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO:7, wherein the receptor specifically binds to a compound that modulatestaste, wherein said compound specifically binds to a sensorytransduction G-protein coupled receptor having the amino acid sequenceset forth in SEQ ID NO: 1, 2, or
 7. 2. The isolated receptor of claim 1,wherein the receptor comprising greater than about 95% amino acidsequence identity to an amino acid sequence of SEQ ID NO:1, SEQ ID NO:2,or SEQ ID NO:7.
 3. The isolated receptor of claim 1, wherein thereceptor has G-protein coupled receptor activity.
 4. The isolatedreceptor of claim 1, wherein the receptor has an amino acid sequence ofSEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:7.
 5. The isolated receptor ofclaim 1, wherein the receptor is from a human, a rat, or a mouse.
 6. Theisolated receptor of claim 1, wherein the receptor is covalently linkedto a heterologous polypeptide, forming a chimeric polypeptide.
 7. Theisolated receptor of claim 6, wherein the chimeric polypeptide hasG-protein coupled receptor activity.